METHODS AND COMPOSITIONS FOR DIAGNOSIS AND TREATMENT OF PARKINSON'S DISEASE AND PARKINSONISM

Statement of Priority

This application claims the benefit, under 35 U.S.C. § 119(e), of U.S. Provisional

Application Serial No. 61/711,608, filed October 9, 2012, the entire contents of which are incorporated by reference herein.

Statement of Government Support

This invention was made with government support under Grant Nos. R01NS064934 and U 8NS082132 awarded by the National Institutes of Health. The government has certain rights in the invention.

Field of the Invention

The present invention is related to methods and compositions for the diagnosis and treatment of Parkinson's disease (PD) and/or parkinsonism in a subject.

Background of the Invention

It is generally recognized that there are inadequate protocols available for detecting disease progression and biochemical pathways associated with PD in clinical populations, and that this lack of effective biomarkers has hindered, and is expected to continue to impede, successful clinical trials for efficacious neuroprotective (e.g., disease -retarding or halting) therapeutics. Genetic studies have identified several proteins linked to late-onset PD and these proteins have provided insight into pathways that might be central to disease. To explore these proteins and pathways of interest, approaches feasible for utilization broadly in clinical trials should be prioritized.

The present invention overcomes previous shortcomings in the art by providing methods and compositions for diagnosing and treating PD and/or parkinsonism in a subject, Summ ary of the Inventio n

The present invention provides a method of diagnosing Parkinson's disease (PD) or parkinsonism in a subject, comprising; obtaining a sample (e.g., a urine sample or

cerebrospinal fluid (CSF) sample) from the subject; detecting the amount of or activity of LRRK2 protein in the sample from the subject; and comparing the amount of or activity of LRR 2 protein in the sample from the subject to the amount of or activity of L3 J K2 protein in a sample of a control, wherein an increase in the amount of or activity of LRRK2 protein in the sample as compared to the control diagnoses PD or parkinsonism in the subject. In some embodiments, this method can further comprise administering to the subject a treatment that decreases the amount of LRRK2 protein and/or inhibits LRRK2 activity in the subject.

Also provided herein is a method of identifying a subject as having an increased likelihood of having or developing Parkinson's disease (PD) or parkinsonism, comprising; obtaining a sample from the subject; detecting the amount of or activity of LRRK2 protein in the sample from the subject; and comparing the amount of or activity of LRRK2 protein in the sample from the subject to the amount of or activity of L RK2 protein in a sample of a control, wherein an increase in the amount of or activity of LRRK2 protein as compared to a control identifies the subject as having an increased likelihood of having or developing PD or parkinsonism.

The present invention also provides a method of identifying a subject as a suitable candidate for treatment that decreases the amount of LRRK2 protein and/or inhibits or reduces LRRK2 protein activity in a subject, comprising: obtaining a sample from the subject; detecting the amount of or activity of LRRK2 protein in the sample from the subject; and comparing the amount of or activity of LRRK2 protein in the sample from the subject to the amount of or activity of LRRK2 protein in a sample of a control, wherein an increase in the amount of or activity of LRRK2 protein as compared to a control indicates that the subject is a suitable candidate for treatment that decreases the amount of LRRK2 protein and/or inhibits or reduces LRR 2 protein activity.

Further provided herein is a method of determining the efficacy in a subject of a treatment for Parkinson's disease (PD) or parkinsonism, comprising: a) measuring the amount or activity of LRRK2 protein in a sample collected from the subject before the treatment; and b) measuring the amount of activity of LRRK2 protein in a sample collected from the subject after the treatment, wherein a decrease in the amount or activity of LRRK2 protein measured after the treatment as compared to before the treatment indicates efficacy of the treatment for PD or parkinsonism. In some embodiments, the treatment can be administration of a substance that decreases the amount o LRRK2 protein and/or inhibits LRRK2 activity in the subject.

The methods of this invention can further comprise the step(s) of treating the subject, e.g., by administering to the subject identified or diagnosed as described above, a substance that decreases LRRK2 protein and/or inhibits LRRK2 activity (e.g., sunitinib). Thus, in further embodiments, the methods of this invention can further comprise the steps of determining efficacy of the treatment by measuring the amount of LRR 2 protein in a sample from a subject before and after administration of a substance that decreases LRR 2 protein and/or inhibits LRRK2 activity, wherein a decrease in the amount of LRRK2 protein measured after administration as compared to before administration indicates efficacy of the treatment.

Brief Description of the Drawings

Figure 1. Discovery of LRRK2 protein enriched in urinary exosomes. A) An exosome pellet was derived from 60 ml of human urine from a healthy young adult, and the pellet was resuspended in PBS and imaged using cryo-E . Pure ~100 nm vesicles were detected. B) The purified exosomes were digested with trypsin with or without exosorne- permeabilizing TRITON-X 100 to determine the localization of LRRK2 in exosomes.

LRRK2 is present on the outside of exosomes, similar to ALIX and other charged multi- vesicular proteins and Rab-type proteins. C) The S 100 fraction, exosome wash buffer, and the exosome pellet were analyzed by western blot and compared to a titration of truncated- recombinant LRRK2 (Invitrogen), The equivalent exosomes from 4 mLs of human urine were loaded on the gel, thus LRRK2 is present at -100 pg/mL in this individual, representing approximately 0.02% of the exosome protein content (this individual had ~500 ng/mL exosomes).

Figure 2. A) Urinary exosome pellets were derived from 6 healthy controls, quantified with BCA analysis, and 1 ug of protein was loaded onto SDS-PAGE gels. The PVDF was split into two, and probed for LRRK2 and TGSIOI (exosome control). B) ECL signal was quantified by an Alpha-innotech HD in replicate runs, and protein content normalized to TGS 101. Large variability in this control population was observed for LRRK2 in contrast with TGSIOI .

Figure 3. A) The cancer drug sunitinib is a potent LRR 2 kinase inhibitor in LRRK2 peptide based kinase assays. B) Rats were treated BID at the indicated dose for two days prior to sacrificing 6 hours post-final drug treatment. Detergent free soluble kidney lysates were generated by dounce homogenization, protein quantified by BCA analysis, and loaded onto immunoblots. Sunitinib treatment destabilized LRRK2 protein at clinically relevant dosages.

Figure 4. Whole-exosome proteomic analysis. A) Commassie gel of purified exosomes compared to a standard of BSA, prior to in-solution mass spectrometry. Abundant band is THP. B) Normalized Spectral Count (NSC) associated with 935 proteins plotted with standard deviations in replicate runs (n=6). The more abundant proteins, as in C) have satisfactory technical error that results in low standard error (indicated). D) ingenuity IPA- analysis indicates the top enriched disease-pathways associated with the exosome protein pool, and neurological-disease linked proteins is the top hit.

Figure S. Localization of LRRK2 to urinary exosomes. A) Human urine was processed into equivalent volumes of supernatant SI 00 (exosome depleted), PI 00 (exosome enriched), or the PI 00 wash buffer. Recombinant LRRK2 is A970-LRRK2 (Invitrogen). B) 10 μg of purified urinary exosomes, treated with or without 200 ng trypsin, with or without 1 % TRITON X-l 00, for 5 min at 37°C. n.s. is non-specific band. C) P 100 urinary exosomes separated across a density gradient and probed for expression of the indicated protein. D) Representative cryo-electron microscopy (EM) image, scale bar is 50 nm. E) LRRK2- fluorescent imaging in urinary exosomes visualized by STED microscopy, scale bar is 500 nm. F) Fluorescent co-labeling of LRRK2 (green) together with TSG101 or CD9. Arrows indicate positive co-localization, scale bar is 2 μπι. G) Blue NativePAGE of LRRK2 protein from purified urinary exosomes, treated with or without 1% TRITON X-l 00. H)

Comparison of relative LRRK2 expression and phospho-serine 935 in 10 g purified urinary exosomes with 10 g of protein lysate from human brain (frontal cortex), or 1 ng of recombinant LRR 2 isolated from HEK-293T cells.

Figure 6. Localization of LRRK2 in the kidney and proteomic characterization of urinary exosomes. A) Coronal rat kidney sections from WT or B) KO animals, stained with LRRK2 antibody ( c41-2, Epitomics) and counterstained with hematoxylin and eosin (H&E). Scale bars for low-magnification images (left panels) are 200 μηι, and 25 μιη for high- magnification (right panels). C) Quantitative TMT-MudPIT results for the most abundant exosome proteins in comparison to known LRRK2 protein interactors. Error bars are S.D. generated by spectral counts from six differential isobaric tags. D) Urinary exosome proteins identified by the Tissue-specific Gene Expression and Regulation database.

Figure 7. LRRK2 exosome release is regulated by 14-3-3. A) Representative cryo- EM image of exosomes purified from HEK-293T cells expressing LRRK2 protein, scale bar is 100 nm. B) HEK-293T cells expressing LRRK2 protein were co-transfected with eGFP, scrambled difopein (a control for difopein, see), or difopein. LRRK2 was

irnmunoprecipitated and exosomes collected from culture media. C, D) Dose-response curves measuring pi 503 autophosphorylation (Alpha Screen signal) in in vitro kinase assays at indicated drug concentrations. IC50 concentrations were calculated through non-linear regression. E) HE -293T cells expressing LRR 2 were treated with the indicated drug (ΙμΜ) or equivalent DMSO concentrations (0.01%) for 36 hours. Cells were harvested into total cell lysates, LRR 2 immunoprecipitated, and exosomes collected.

Figure 8. LRRK2 cytoplasmic localization is regulated by 14-3-3. A-H)

Representative confocal images of HEK-293T cells expressing mKate2-tagged LRRK2 (N- terminal tag), with cells treated with the indicated drug and/or co-transfected with the indicated construct. Drugs were used at 1 μΜ concentration for 36 hours.

Figure 9. Lack of effect of LRRK2 mutations and lack of localization with a- synuclein in exosomes. A) Immunofluorescence co-localization of mKate2-LRRK2 with the MVB marker TSG101 using confocal immunofluorescence. A representative cell is shown. B) LRRK2-myc, with the indicated mutation, was transiently transfected into HEK-293T cells and exosomes and cytosol lysates collected 48 hours after transfection. C) Purified exosomes from HEK-293T ceils transfected with mKate2-LRRK2 and a-synuclein. No overlapping signal could be observed. All data are representative of at least three

independent experiments, scale bars represent ΙΟμ ι.

Figure 10. Characterization of exosomal LRRK2 in clinical populations. A)

Exosomes were isolated from urine of PD and control patients, and lysates were analyzed by western blot in quadruplicate independent runs. The "pool" value is a sample comprised of 10% of each exosome lysate of the 20 PD and 15 control individuals mixed together, allowing comparison of samples analyzed on different gels. B-D) Plots showing relative LRRK2 expression normalized to TSG101 expression; data points of each subject are the mean of four independent runs, with removal of subject outliers. Error bars are S.E.M. and the horizontal line is the cohort mean. E) Titration of an outlier PD affected subject, in comparison to the cohort geometric mean (GM) sample that has average LRRK2 and TSG101 expression. F) Clinical summary of the PD affected case analyzed in panel E. G) Urinary exosomes collected from a neurological ly normal individual over the course of 1 week, and analyzed for LRRK2 and TSG101 expression.

Figure 11. LRR 2 exosome secretion by macrophages. A) Macrophage cell line Raw 264.7 cells were treated with or without LPS (lOOng/ml) and exosomes and cell cytosols collected 24 hours later. B) Thioglycollate collected primary macrophages were isolated from WT or G2019S-BAC mice and exosomes and cytosol lysates collected 24 hours post- LPS transduction. C) Quantification of exosomal LRRK2 from panel B. D, E) Confocal images of thioglycollate collected peripheral primary macrophages isolated from LRRK2 KO or LRRK2 BAC-transgenic mice were stained for LRRK2 protein (antibody c41-2) or TSGIOI. LPS (100 ng/ml) was used for 24 hours prior to fixing cells. Representative images are shown; results were typical of three independent experiments. Scale bars are 10 μιη for all panels.

Figure 12. LRRK2 exosome secretion by neurons and in the CSF. A) Representative coronal section through the striatum of either WT or LRR 2 O mice. Arrows indicate LRRK2 positive cells in striosome-like patches present in the striatum and juxtaposed to lateral ventricles. Scale bar is 0.2 mm. B) Representative cryo-EM image of exosomes isolated from human CSF. Scale bar is 50 nm. C) Western blot quantification of LRRK2 in purified CSF exosomes (A970-rLRR 2, Invitrogen). CSF loaded onto the lane represents exosomes from the equivalent of 1 ml of CSF. D, E) Primary cortical neurons transduced with LRRK2 adenovirus were lysed 48 hours post-transduction and exosomes purified.

Quantification of relative G2019S and WT LRRK2 in exosomes secreted by neurons, normalized to cytosolic LRRK2 levels. F) Primary neurons cultured for 10 days in vitro, transduced with low concentrations (-0.1 multiplicity of infection) of WT-LRRK2 virus at 5 days in vitro, were stained for LRRK2 protein (antibody N241 ) or TSG101. Arrow heads show co-labeled vesicles, dendrites were identified by intense MAP2 labeling. White scale bars are 5 μηι.

Figure 13. Proposed model for LRRK2 exosomal release. Detailed Description of the Invention

In the following detailed description, embodiments of the present invention are described in detail to enable practice of the invention. Although the invention is described with reference to these specific embodiments, it should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. All publications cited herein are incorporated by reference in their entireties for their teachings.

The invention includes numerous alternatives, modifications, and equivalents as will become apparent from consideration of the following detailed description.

As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. For example, "a" cell can mean one cell or a plurality of cells.

It will be understood that although the terms "first," "second," "third," "a)," "b) and "c)," etc. may be used herein to describe various elements of the invention should not be limited by these terms. These terms are only used to distinguish one element of the invention from another. Thus, a first element discussed below could be termed a element aspect, and similarly, a third without departing from the teachings of the present invention. Thus, the terms "first," "second," "third," "a)," '¾)," and "c)," etc. are not intended to necessarily convey a sequence or other hierarchy to the associated elements but are used for

identification purposes only. The sequence of operations (or steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of

combinations when interpreted in the alternative ("or").

Furthermore, the term "about," as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1 % of the specified amount.

As used herein, the transitional phrase "consisting essentially of means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 21 11 , 03.

A "sample" or "biological sample" of this invention can be any biological material, such as a biological fluid (e.g., urine, cerebrospinal fluid, blood, serum, plasma, saliva, joint fluid, semen, washings, etc.), an extract from a cell, an extracellular matrix isolated from a cell, a cell (in solution or bound to a solid support), a tissue, a tissue homogenate, and the like as are well known in the art.

As used herein, "one or more" can mean one, two, three, four, five, six, seven, eight, nine, ten or more, up to any number.

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

As used herein, the term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. The term "nonhuman animals" includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, pig, cat, horse, cow, chickens, amphibians, reptiles, rodents (e.g., mice, rats, etc.) and the like. In particular embodiments, the subject of this invention is a human subject. A "subject in need thereof or "a subject in need of is a subject known to have, or is suspected of having or developing PD and/or parkinsonism and/or a PD-associated disorder or PD-associated condition of this invention or is at risk of having or developing PD and/or parkinsomism and/or a PD-associated disorder or PD-associated condition as described herein.

As used herein, Parkinson's disease refers to a well known and well characterized neurodegenerative disorder. "Parkinsonism" refers to a condition that results in a

combination of movement abnormalities including tremors, rigidity, slow movements, and slurred or impaired speech. As is known in the art, not everyone who has parkinsonism has Parkinson's disease.

The term "administering" or "administered" as used herein is meant to include topical, parenteral and/or oral administration, all of which are described herein. Parenteral administration includes, without limitation, intravenous, subcutaneous and/or intramuscular administration (e.g., skeletal muscle or cardiac muscle administration). In the methods of this invention, the compound or composition of this invention may be administered alone and/or simultaneously with one or more other compounds. In some embodiments, the compounds may be administered sequentially, in any order. It will be appreciated that the actual method and order of administration will vary according to, inter alia, the particular preparation of compound(s) being utilized, and the particular formulation(s) of the one or more other compounds being utilized. The optimal method and order of administration of the compounds of the invention for a given set of conditions can be ascertained by those skilled in the art using conventional techniques and in view of the information set out herein.

The term "administering" or "administered" also refers, without limitation, to oral, sublingual, buccal, transnasal, transdermal, rectal, intramuscular, intravenous, intraarterial (intracoronary), intraventricular, intrathecal, and subcutaneous routes. In accordance with good clinical practice, the instant compounds can be administered at a dose that will produce effective beneficial effects without causing undue harmful or untoward side effects, i.e., the benefits associated with administration outweigh the detrimental effects.

Also as used herein, the terms "treat," "treating" or "treatment" refer to any type of action that imparts a modulating effect, which, for example, can be a beneficial and/or therapeutic effect, to a subject afflicted with a condition, disorder, disease or illness, including, for example, improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disorder, disease or illness, delay of the onset of the disease, disorder, or illness, and/or change in clinical parameters of the condition, disorder, disease or illness, etc., as would be well known in the art.

An "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition of this invention that is sufficient to produce a desired effect, which can be a therapeutic and/or beneficial effect. The effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an effective amount or therapeutically effective amount in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation. (See, for example, Remington, The Science and Practice of Pharmacy (latest edition)).

As used herein, the term "ameliorate" refers to the ability to make better, or more tolerable, a condition, disorders and/or symptom. The term "prevent" refers to the ability to keep a condition, a reaction, a disorder and/or symptom from happening or existing or developing.

"LRRK2" refers to the Leucine-Reach Repeat Kinase 2 protein (NCBI Accession. No. NP_490980.3 in Homo sapiens, NCBI Accession No. NPJ ⁾ 01178718.1 in Rattus norvegicus, NCBI Accession No. NP_080006.3 in Mus musculus). Missense mutations in the LRRK2 gene cause late-onset Parkinson's disease (PD) in both autosomal-dominant disease transmitting families, and in sporadic late-onset disease populations. In addition to linkage to PD through genome-wide association studies, LRRK2 genetic variants impose susceptibility risks to inflammation-linked diseases that include Crohn's disease and mycobacterial infection. The LRRK2 gene encodes a protein with a unique multi-domain composition, including functional GTPase and protein kinase domains, and missense mutations that cause PD alter these enzymatic activities. Understanding the function of LRRK2 can provide insight into pathogenic mechanisms as well as determine particular targets for disease- modifying therapeutics.

The present invention is based on the unexpected discovery that proteins linked to or associated with PD and parkinsonism can be detected and quantified from exosomes derived from human urine samples that can be obtained, e.g., in clinical settings. Previously, emphasis has been placed on the use of CSF, which has been challenging because of limited patient participation, especially controls, the expense of the procedure, and the fact that only small amounts of CSF can be drawn and the resultant fluid is very dilute in proteins (including exosomes) and other markers of interest, and CSF is susceptible to variable contamination of blood cells during the procedure.

Thus, one aspect of the present invention is based on the discovery that proteins linked to PD, such as, e.g., LRRK2 and DJ-1 (linked to a recessive form of Parkinsonism) can be detected and quantitatively measured in exosomes derived from human urine fractions. These exosomes can be easily obtained in clinical settings, as only urine samples are required, and in the present invention, protocols have been developed to measure LRRK2 and DJ-1 expression from urine samples. In addition, technology has been developed to quantitatively measure 935 other proteins using an innovative multi-plex mass spectrometry proteomic platform that can scale to large clinical populations. Of these 935 proteins that can be detected in control urinary exosome fractions, 140 of these proteins are linked to neurological diseases by network and pathway analysis, and neurological disease is the top enriched disease/disorder (p=6.85 ^~5 ) of the detected exosome proteins.

LRRK2 expression in mammals is widely distributed in many cell types but particularly enriched in the kidney and in activated macrophages of the innate immune system. In the brain, LRRK2 expression is relatively modest and includes medium spiny neurons in the striatum that form striosomes. On a subcellular level, LRRK2 associates with a number of vesicle types and to intraluminal vesicles within multivesicular bodies (MVBs). Assignment of LRRK2 function within the endocytic pathway has been suggested, with recent evidence for action in retrograde vesicle trafficking from endosomes to the trans-Golgi network, and for modifying chaperone-mediated autophagy. Over-expression of the late- endosome GTPase Rab7 and related Rab7 isoforms rescue LRRK2-mediated phenotypes in neurons, while RNAi knockdown of ArfGAPl, a protein associated with vesicle biogenesis, also rescues LRRK2 phenotypes in neurons.

Elevated LRR 2 kinase activity is linked to PD susceptibility since the most common pathogenic LRRK2 mutation G2019S in humans increases kinase activity approximately 3- fold in most assays, and overall LRR.K2 subcellular localization has been described as exquisitely sensitive to acute LRRK2 kinase inhibition. LRRK2 interaction with 14-3-3 proteins appears to be one of the main modulators of LRRK2 subcellular localization, consistent with the notion that 14-3-3 isoforms principally function by modulating cell localization of binding partners. However, there are no known physiological roles described for the 14-3-3 LRRK2 interaction or impact that 14-3-3 mediated localization may have on LRR 2 functionality, particularly within the endocytic pathway. LRRK2 is secreted from intra-luminal vesicles from MVBs (i.e., exosomes) from a variety of cells where LRRK2 is natively expressed, including cells in the kidney, brain, and immune system. LRRK2 can be readily detected through purification of exosomes from urine or cerebral spinal fluid (CSF) in clinical populations, or through exosomes from cell culture media. It has also been found that a major role for the 14-3-3/L RK2 interaction may be the regulation of LRRK2 association with late-endosomes and uptake into MVBs with subsequent extracellular release of LRRK2 protein via exosome secretion. These studies have elucidated a new component to LRRK2 functionality in the endocytic pathway and open the door for LRRK2-targeted biomarker and clinical trial studies through characterization of a convenient source for LRRK2 protein (e.g., urine and CSF).

Thus, in one embodiment, the present invention provides a method of diagnosing PD in a subject, comprising detecting an increase in the amount or activity of LRRK2 protein in a sample from the subject as compared to a control, thereby diagnosing PD in the subject. Although not particularly limited, one embodiment of the method comprises detection of LRRK2 protein from a sample obtained from urine or CSF. In another embodiment, the urine sample is collected fresh, i.e., has not been frozen and is processed the same day as collection. For example, if collected in the morning, the urine sample is processed in the afternoon. In some embodiments, prior to processing, the sample is stored at 4°C for no longer than about two hours following collection. In another embodiment, processing comprises isolation of exosomes from the sample. In a further embodiment, the method comprises detection of LRRK2 protein found in exosomes isolated from the sample,

Although the cellular source of the exosomes in the sample is not particularly limited, in one embodiment, the exosomes isolated from a urine sample are derived from the kidney.

Isolation of exosomes from a sample for analysis may take place according to any protocol for isolating exosomes that will be understood by one of skill in the art.

Detection of the amount of LRRK2 protein in a sample can be carried out by any technique using any standard protocol that will be appreciated by one of skill in the art. In one embodiment of the invention, detection of LRRK2 protein may take place using immunohistochemistry or immunofluorescence. In another embodiment of the invention, detection of LRRK2 protein can be achieved using cryo-electron microscopy (cryo-EM) or standard emission depletion (STED) microscopy. In still another embodiment, detection of LRRK2 protein may employ western blotting assays.

The protocol for detecting the activity of LRRK2 protein in a sample can be carried out by any known procedure to assay for LR-RK2 activity that is known to one of skill in the art. LRRK2 is a multi -domain protein with multiple activities. In one embodiment, activity of LRRK2 protein is detected by measuring kinase activity of LRRK2 protein. Kinase assays of LRRK2 protein may be performed according to any standard protocol for measuring kinase activity that is known to one of skill in the art.

Determining whether the subject has PD is made by comparing the amount or activity of LRRK2 protein of the sample to the amount or activity of LRRK2 protein detected in a control sample, wherein an increased or greater amount or activity of LR K2 protein in the sample from the subject as compared to the amount or activity of LRRK2 protein in the control sample is indicative of the subject having PD. A control is, for example, a sample from a normal subject, or subjects, who does not or do not have PD.

The significance of the comparison between subject sample and control sample may be determined through statistical analysis. General analyses may be accomplished, for example, using SAS version 9.3, R, StatXact, GeneSring, Partek, and Ingenuity Pathway Analysis (IP A) software packages, depending on the test. For categorical variables (e.g., PD affected/unaffected, sunitinib treated/untreated, LRRK2 high/low expression, etc.), estimates of proportions and confidence intervals can be calculated. For group comparisons of proportions, the Chi-square or Fisher's exact test can be used as appropriate. A paired comparison of proportions can be analyzed using the McNemar's test. The logistic regression model can be used for group comparisons of proportions when covariate adjustments are desired. Model building using different selection procedures can be performed to determine the significant factors in the model. Methods of cross-validation can be employed when feasible. Likelihood ratio tests, R ² and analysis of residuals can be performed to assess the adequacy of the resulting model.

Identifying increased likelihood of having or developing PD

In another embodiment, the present invention provides a method of identifying a subject as having an increased likelihood of having or developing PD, comprising detecting an increase in the amount or activity of LRRK2 protein in a sample from the subject as compared to a control. As will be appreciated by one of skill in the art the protocols for obtaining a sample, detecting the amount of and/or detecting the activity of LRRK2 protein can be the same as the protocols set forth above for the diagnosis of PD according to the present invention,

A determination of whether the subject has an increased likelihood of having or developing PD is made by comparing the amount or activity of LRRK2 protein of the sample from the subject to the amount or activity of LRR 2 protein detected in a control sample, wherein an increased or greater amount or activity of LRRK2 protein in the sample from the subject as compared to the amount or activity of L K2 protein in the control sample is indicative of the subject having an increased likelihood of having or developing PD. A control is, for example, a sample from a normal subject or subjects, who does not or do not have PD. The significance of the comparison between subject sample and control sample can be determined through statistical analysis as set forth above for the diagnosis of PD according to the present invention.

Identifying suitable candidates for treatment

In a further embodiment, the present invention provides a method of identifying a subject as a suitable candidate for treatment with a substance that decreases the amount of L RK2 protein in the subject and/or inhibits or reduces LPJRK2 activity in the subject, comprising detecting an increase in the amount or activity of LRRK2 protein in a sample from a subject as compared to a control sample. In one embodiment, the substance that decreases the amount of LR K2 protein in the subject and/or inhibits or reduces LRRK2 activity in the subject is a kinase inhibitor. In one embodiment, the kinase inhibitor can be sunitinib. As will be appreciated by one of skill in the art, the protocols for obtaining a sample, detection of the amount of and/or the activity of LRRK2 protein can be done according to the same protocols set forth above for diagnosing PD or parkinsonism or for determining the likelihood of having or developing PD or parkinsonism according to the present invention.

A determination of whether the subject is a candidate for treatment with a substance that decreases the amount of L RK2 protein in the subject and/or inhibits or reduces LRRK2 activity in the subject is made by comparing the amount of or activity of LRRK2 protein of the sample from the subject to the amount of or activity of LRRK2 protein detected in a control sample, wherein an increased or greater amount or activity of LRJRK2 protein in the sample from the subject as compared to the amount or activity of LRRK2 protein in the control sample identifies the subject as a candidate for treatment with a substance that decreases the amount of LRR 2 protein in the subject and/or inhibits or reduces LRRK2 activity in the subject. A control sample is, for example, a sample from a normal subject or subjects, who does not or do not have PD. The significance of the comparison between subject sample and control sample can be determined through statistical analyses as set forth above for the diagnosis of PD or for determining the likelihood for having or developing PD according to the present invention. Methods of treatment,

In an additional embodiment, the present invention provides a method of treating a subject having PD, such as a subject identified by the method described above, comprising to the subject an effective amount of a substance that decreases the amount of LRRK2 protein and/or inhibits LRRK2 activity in the subject. In one embodiment, the substance that decreases the amount of LRR 2 protein in the subject and/or inhibits or reduces LRRK2 activity in the subject is a kinase inhibitor. In a further embodiment, the kinase inhibitor is sunitinib. In yet a further embodiment, the efficacy of the treatment is monitored by measuring the amount or activity of LRRK2 protein in a sample from the subject before administration and after administration of the substance that decreases the amount of LRRK2 protein in the subject and/or inhibits or reduces LRRK2 activity in the subject, wherein a decrease in the amount and/or of LRRK2 protein in the subject after administration of the substance indicates efficacy of the treatment. As will be appreciated by one of skill in the art the protocols for obtaining, detecting the amount of and/or detecting the activity of LRRK2 protein can be the same as the protocols set forth above for diagnosing PD, determining the likelihood of having or developing PD, or identifying whether a subject is a candidate for treatment with a substance that decreases the amount of LRRK2 protein in the subject and/or inhibits or reduces L K2 protein activity in the subject according to the present invention.

It is indicated that total LRRK2 as well as L RK2 phosphorylated at amino acid 1292 of the LRRK2 protein will be reduced in individuals taking a LRR 2 kinase inhibitor, or other therapeutics used to treat Parkinson's disease relevant to LRRK2. This has utility in terms of clinical trials. For example, if signal remains high for LRR 2 and pi 292 in an individual taking a kinase inhibitor or other therapeutic, more drug may be needed for that individual to achieve inhibition (reduced exosome LRR 2 protein, or reduced i 292). For a Phase I clinical trial, reduced exosome LRRK2 signal may be an achievable endpoint for success of the therapeutic. For Phase II and beyond, the test can be used for individualized treatment, such that some patients may respond to low levels of drug with complete LRRK2 removal from exosome, and thus have minimal off-target and drug side effects from taking lower amounts of drug, whereas some patients may need higher amounts of drug to inhibit LRRK2 and remove LRRK2 signal from the assay, and these higher amounts would be needed to see therapeutic benefit (i.e., neuroprotection in Parkinson's disease, or other related diseases). Thus, the methods of this invention can include additional steps such as modulating (i.e., increasing or decreasing) the dose of a kinase inhibitor or other therapeutic on the basis of a change in the amount of LRRK2 detected in a sample from a subject. For PD diagnosis, the pi 292 LRRK2 levels, and/or total LRRK2 protein levels, can identify individuals at increased risk of developing Parkinson's disease. These individuals would be suitable candidates to receive neuroprotective therapeutics to stave off the onset of Parkinson's disease. Mutations in the LRR 2 protein cause PD, but only in about 50% of patients with any given LRRK2 mutation. Measurement of pi 292 and/or total LRRK2 in urinary exosomes can show that the affected (manifesting) patients have higher levels of signal in the assay. Thus, known LRRK2 mutation carriers may want to monitor their levels of active LRR 2 ( i 292 lrrk2) and total LRRK2 to assess risk of development of PD, and possibly enroll in clinical trials or take other pro-active measures, should the test reveal heightened risk for PD.

Detection of LRRK2 in urinary exosomes can correlate well with other markers in blood and urine in disease, and provide a biochemical link and/or the discovery of additional biomarkers that are easier to assay and quantify than LRRK2 is; post-LRR 2 detection, levels of hundreds of other proteins and chemicals will be evaluated to shed light on mechanisms important in PD and other diseases or physiological processes like aging.

Proteomic Analysis

In yet another embodiment, the present invention provides a method of screening for substances/molecules/proteins related to PD. In one embodiment, the analysis is performed on exosomes, wherein the exosomes have been isolated from either urine or cerebral spinal fluid (CSF). In another embodiment, the analysis examines modulators of the activity of

LRRK2 protein. In another embodiment, the analysis examines modulators of the activity of DJ-1. These screening methods can be carried out according to the protocols described herein. EXAMPLE 1

Exosomes in neurodegeneration and PD. Exosomes are relatively recently discovered structures that are small -100 nm average vesicles encapsulated intra-cellularly in the cytosolic of numerous cell types within multi-vesicular bodies (MVB). Upon MVB binding to the outer membrane, some or all of the exosomes are released to the extracellular environment, and these exosomes contain a rich array of proteins, lipids, and RNA. Released exosomes can be readily purified and visualized in native conformation (aqueous, without fixation or processing) by a technique known as Cryo-Electron Microscopy (Figure 1A). It is believed that exosomes are an evolutionarily conserved structure for intercellular

communication and also the principle mechanism of moving most proteins and RNAs that can be trafficked between cells. Many types of cells, including neurons and glia, are able to endocytize exosomes and functionally incorporate the cargo. The protein composition of exosomes is speculated to generally reflect much of the protein constituency of the originating cell's cytosol, based on the thousands of proteins identified from a number of highly purified exosome sources in humans (see www.exocarta.org). Due to the accessibility of exosomes in human fluids and early-linkages to biological processes, it is considered that exosomes may hold tremendous value as biomarkers for human disease.

In neurodegeneration, exosomes have been proposed as the primary vehicles for transfer of toxic proteins. Exosomes can transmit prion diseases through the trafficking of PrP-Sc. In models of Alzheimer's disease, trafficking of Αβ and components of APP are in part accomplished via exosomes. In cell models of ALS, mutant SOD-1 can traffic through exosomes and aggregate in the host cell. In PD, primary neuronal cultures secrete both tau and alpha-synuclein monomers and aggregates in exosomes. In fact, alpha-synuclein is thought to be expressed solely in neurons but aggregates can be found in glia cell types in MSA and other neurological diseases, and exosome transport of alpha-synuclein is the likely process for this to occur. Mutations in VPS35 cause late-onset PD and VPS35 is involved in retrograde transport of endosomes to the Golgi network, and possibly endosomes to MVB critical for exosome formation. Thus, the proteins believed to be at the heart of most neurodegenerative disorders are associated with exosomes in some manner. However, the specific role of exosomes in disease etiology is not clear.

L RK2 and PD-proteins in urinary exosomes. The interest in exosomes originated from localization work with the LRRK2 protein. LRRK2 was discovered in late-onset PD kindreds and subsequently identified as the most common genetic cause of late-onset PD, where pathogenic mutations are causing disease in 20-30% of late-onset cases in some populations. In most Caucasian populations, pathogenic L RK2 mutations are present at ~1- 5%, and common variation in the L RK2 gene associates with LRR 2 susceptibility in large GWAS meta-analyses. However, whether LRRK2 expression or activity is abnormal in PD without LRPvK2 mutations (e.g., the vast majority) is unknown. Studies to address L RK2 and other proteins of interest in PD have been difficult since the neurodegenerative condition irreversibly alters cell composition and proportions in affected areas, leaving behind cells that are resistant, or sick, or reactive to the inflammatory process (e.g., L RK2 -filled microglia), and no easy way to distinguish between these cells for most biochemical approaches. In addition, post-mortem brain material inherently has variables such as pH, delay to collection (PMI), and time spent in storage, that greatly influence most metrics associated with proteins. We have been interested in normal LRRK2 expression and localization and found that R 2 localizes to vesicular structures in cells that normally express LRRK2. The LRRK2 GTPase domain is most similar to human Rab-vesicular sorting proteins (BLAST analysis), and Rab proteins are among the more abundant species on exosomes and directly participate in vesicular trafficking in cells. EM studies have also revealed LRRK2 association with MVBs (i.e., exosome precursors). A database search (www.exocarta.com) showed that LRRK.2 has been detected in exosomes in mass spectrometry studies. Moreover, several other PD proteins in addition to LRR 2 were detected in exosomes isolated from human urine. In addition to LRRK2, VPS35 protein and DJ-1 protein are present. To confirm these findings, we developed our own protocol to isolate human urine exosome fractions, verified their purity by cryo-EM and immunoblots, and found that LRRK2 is readily detectable by immunoblot (see Figure 1).

A whole-proteomic approach to measure proteins in human urinary exosomes was developed and used to detect 140 proteins implicated in neurological disease (see Figure 4). Despite the robustness of this approach, LRRK2 could not be reliably detected with our multi-plex TMT-MudPIT approach. In addition, although both tau and alpha-synuclein are clearly linked to PD and are packaged into exosomes, we were unable to detect signals for either proteins in urinary exosomes by western blot or mass spectrometry. Based on these studies, it is proposed to utilize both unbiased proteomic screening that can quantify 935 proteins in human urinary exosomes including PD-associated proteins, and also a targeted approach using western blots to quantify LRRK2 specifically because of the strong link to late-onset PD.

Exosome analysis in PD: While it would be ideal to directly analyze susceptible brain tissue in PD cases early in disease, later in disease, and longitudinally during a clinical trial for neuroprotective therapeutics, brain tissue specimens are obviously limited to postmortem collection. Novel imaging approaches are very promising non-invasive procedures, but these techniques have not yet been able to directly measure proteins or biochemical pathways that may underlie neurochemical changes. Emphasis has been placed in recent years on analyzing CSF, which has been challenging because of limited patient participation (especially controls), the expense of the procedure, and that only small amounts of CSF can be drawn and the resultant fluid is very dilute in proteins (including exosomes) and other markers of interest, and CSF is susceptible to variable contamination of blood cells during the procedure. In contrast, urine is easily collected and analyzed from patients and controls alike. Immediately after collection, we introduce a robust 20x stabilization buffer that normalizes out variation such as pH and protease activity. Although proteins of interest, for example proteins linked to the pathology or genetics of PD, are also very dilute (e.g., LRRK2 is ~100pg/mL, Figure 1), hundreds of mLs (100 mL=3.38 ounces) can be obtained from most patients, even those with trouble urinating, and processed rapidly, in stark contrast to CSF.

The abundance of proteins already known to be linked to late-onset PD can be

quantitatively measured from these exosomes. It is reasonable to consider that peripheral expression in exosomes might correlate to expression in PD-susceptible tissue. It is also possible that urinary exosomes directly sample proteins from a variety of organs, including the brain.

On a network analysis, 140 proteins that have been detected with our proteomic analysis have been linked to neurodegenerative processes. For example, LRRK2 and alpha-synuclein- interacting proteins 14-3-3, HSP90, and HSP70, ERM/Radixin, are detectable. Each of these can be quantified using very standardized and controlled conditions of collection and processing afforded by the non-invasiveness of the urine collection procedure. In addition, given the combination of both a targeted and unbiased proteomic approach, we have the opportunity to discover new protein biomarker associations with PD involving proteins that have not been previously considered in pathogenesis.

Proteins can be measured in a longitudinal manner in clinical populations, and can potentially be used to assess the effects of treatments on the intended targets. For example, during a clinical trial of LRRJ .2 inhibitors, it would be possible to directly assay changes in LRRK2 expression and phosphorylation. This analysis would provide vital information. Also, we have the opportunity to be well powered to detect changes associated with early PD (Hoehn and Yahr stage 1 -2), which is not easily done with post-mortem tissue.

As set forth herein, the payoff of a PD biomarker in urinary exosomes would be incredibly large, and the biomarker could be widely implemented by most centers. With the finding of protein(s) of interest, cheap ELISA and western blot approaches can be developed to follow up on mass spectrometry results. Based on the data included, at a minimum the development of urinary exosomes as a biomarker for LRRK2 target engagement in LRRK2 clinical trials is very likely to succeed, and this is essential for de-risking LRRK2 inhibitors for pharmaceutical development and phase experiments. The identification of urinary exosome biomarkers related to the state or progression of the disease has potential for very high reward. Exosome analysis in PD cases and controls. To our knowledge, there has not been a single effort attempted to analyze exosomes derived from PD patients and controls, isolated directly from either CSF, urine, or plasma, despite the fact that proteins associated with PD are abundant in these structures and these fluids. We propose to tackle urine exosomes first, since it is easy to collect urinary exosomes from large clinical populations. We plan to move on to CSF and plasma in future projects that will allow us to look at other proteins like tau and alpha-synuclein.

Multi-plex whole-exosome proteomic approaches, A relatively low cost technique for rapidly and quantitatively assessing nearly a thousand proteins from exosome preparations has been developed. The usage of a tandem-mass-Tag multidimensional protein

identification technology (TMT-MudPIT) approach together with novel bioinformatic approaches has never been described before in exosome research or PD research, and is considered absolute state-of-the-art in biomarker discovery. This approach can be widely implemented in a number of human diseases in the search for biomarkers.

Understanding LRRK2 and PD-linked genes in late-onset PD. Missense mutations in the LRRK2 gene that are predicted to enhance GTP-binding (e.g., R1441C, Y1699C) or kinase activity (e.g., G2019S) in the encoded protein may induce gain-of-function activity. As it appears that gain or loss of function mutations associate with PD in LRRK2, it is a possibility that enhanced LRRK2 expression may represent a risk factor for PD susceptibility. Similarly, reduced levels of DJ-1 may be a risk factor, potentially in concert with LRRK2 expression. This hypothesis has been extremely difficult to test in clinical populations for the reasons mentioned above concerning post-mortem tissue. The embodiments of this invention have the developed technology and sample availability to quantitatively measure the abundance of these proteins in PD versus control samples, and therefore can test, in relevant samples sizes, whether there is evidence of abnormal expression.

Development of a pharmacodynamic biomarker for LRRK2 kinase activity in patients. Recent clinical trials for neuroprotective kinase inhibitors in PD, such as the double-blind-placebo -controlled, multi-dose, 65 center phase 2/3 clinical trial of CEP- 1347 (the PRECEPT trial), ended with very uncertain conclusions. Although CEP- 1347 had efficacy in several animal models, the human trial was negative. A major limitation of the human study is that it was not possible to determine whether dosage of CEP- 1347 used actually achieved the intended inhibition of the target enzyme, mixed-lineage kinases 1/3. This lack of evidence for "on-target" efficacy, and lack in general of data on target engagement is a major barrier to progress in human subjects research. Because there is evidence that LRRK2 knockout animals are resistant to the neurodegenerative effects of alpha-synuclein over-expression, and mutations may induce increases in kinase activity that cause iate-onset PD, inhibition of LR K2 kinase activity may provide neuroprotective benefit. This invention provides a plan to develop and validate technology to test target engagement in LRRK2 protein isolated from kidney exosomes. Specifically, we expect to see vastly diminished levels of LRRK2 phosphorylation and lower LRR 2 expression in urinary exosomes when LRRK2 is inhibited by sunitinib exposure. This expectation draws from background studies that demonstrate that a kinase-inactivating knock-in mutation in mice greatly destabilize and reduces the expression of LRRK2, and that LRRK2 readily aggregates in transfected cells lines when exposed to a LRRK2 inhibitor, thereby possibly preventing release into soluble fractions and exosomes. We also show greatly reduced

expression in the soluble fraction of rat kidneys treated with sunitinib (Figure 3B), and have demonstrated in cell lines that LRRK2 moves to insoluble fractions when inhibited. While we acknowledge that even if we are successful in showing peripheral engagement of LRRK2, this does not necessarily suggest LRRK2 engagement in the brain. However, for the best molecules that are blood-brain-barrier transparent, peripheral inhibition would be expected to closely mimic inhibition in the brain. Our approach can also be rapidly applied to CSF exosome purifications that can be complemented to larger studies involving urinary exosomes.

Patient inclusion criteria. Inclusion criteria will be based on UK Brain Bank criteria for the clinical diagnosis of PD, and will require: 1) the presence of bradykinesia and either rest tremor or rigidity; 2) asymmetric onset; 3) progressive motor symptoms 4) age at onset 35-85 years.

Sunitinib cohort criteria. Neurological ly normal patients, without history of PD, currently undergoing sunitinib treatment for cancer. Urine will be collected 4-hours (peak serum drug levels in most people) after their usual AM dosage of drug (cancers include pheochromocytoma, carcinoid, or renal cancer with still normal kidney function (GFR >60)).

Control inclusion criteria. Ages of between 35-85 years, a lack of PD in first-degree blood relatives, and a lack of positive responses on more than 3 items on our PD Screening Questionnaire.

Exclusion criteria for all subjects. Include atypical features indicative of a

Parkinson-Plus disorder (Progressive Supranuclear Palsy (PSP), Multiple System Atrophy (MSA), Corticobasal Degeneration (CBD)) including cerebellar signs, supranuclear gaze palsy, apraxia and other cortical signs, or prominent autonomic failure, neuroleptic treatment at time of onset of parkinsonism, active treatment with a neuroleptic at time of study entry, history of repeated strokes with stepwise progression of parkinsonism, history of repeated head injury, history of definite encephalitis, prominent gait imbalance early in the course (< 5 years), dementia, known severe anemia (hematocrit <30), history of kidney disease and/or current or past glomerular filtration rate (GFR <60) possibly indicative of kidney disease, or a serious comorbidity that may interfere with participation in the study.

Databasing Clinical Phenofypes. Clinical assessments are conducted by board certified neurologists with subspecialty training in movement disorders, and nurse-clinicians with specialized training in PD. Phenotype collection meets core and relevant requirements of the PDBP clinical data elements. Cases and controls have all phenotype data uploaded into a database (into a Microsoft Access platform run on a SQL server available to this project). All individuals associated with the study have access to these data. Data on all subjects further conform to the NINDS Common Data Elements toolkit to ensure uniformity of data collection, optimize data analyses and data mining

(www.commondataelements.ninds.nih.gov). Demographics include gender, age, race, ethnicity, education, PD Medical History Onset and nature of PD symptoms, degree of diagnostic certainty, PD Family History Relatives and family members with PD, RFQ-U Height and Weight Height and weight data, for use in covariate analyses, non-prescribed drug use, vitamins and nutriceuticals for covariate analyses, Movement Disorder Society- Unified Parkinson's disease Rating Scale, the Hoehn and Yahr rating scale, and Parkinson's disease Medication Log Estimated dose and duration of therapy with levodopa and other PD medication, Non-Parkinson's disease Medication, Log Estimate dose and duration for non-PD medications. Notably, there will be no permanent sample banking in this study as all clinical samples are completely consumed in the proposed analyses.

Sample Collection. 4-6 samples on average are be collected per week. Urine samples are collected between 8:00AM and 10:00AM during routine clinic hours to avoid possible diurnal variations, and these samples are processed into exosome fractions that afternoon. In general, control samples are collected from spouses or significant others. PD is more common in males, and we anticipate that the study population will have a male:female ratio of 2:1, reflecting the gender specific prevalence of the disorder. Control recruitment is monitored to achieve matching of ages and genders. Based on our estimations of ~0.5 ug of exosome protein per mL of urine (this number is also well-established in the literature), we require at least 70 mL (i.e., 2.3 oz) of urine for enrollment. Even those individuals with trouble urinating can produce this low quantity. When a specimen is collected in the clinic, a small fraction is sent for standard urinalysis (~20 mL) and the rest immediately combined with our 20x urine-exosome stabilization solution in 50 mL aiiquots. The stabilization buffer has been optimized so that no adverse effects on exosome stability are observed up to our longest time point analyzed of 8 hours post aliquoting. Protocols to normalize the effects of variable pH, ionic concentration, active proteases and phosphatases have been previously created, and each sample stored at 4°C for a maximum of 2 hours before processing. The composition of the 20x supplement is 1M Tris pH 7.4, 20mM AEBSF, 40mM EDTA, 1 OOmM b-glycerol-phosphate, 20uM pepstatin A, 20uM E-64.

Large deleterious effects on exosome quantity and quality have been observed if urine samples are frozen or biobanked in a standard manner. The presumed reason for this is that the abundant THP protein in urine tends to fibrilize during the freezing process and these fibrils recruit exosomes that are lost in required low speed spins. One work-around that has been implemented in other laboratories is to include high concentrations of DTT (dithiothreitol). This is not optimal for our goals because DTT will remove outer-exosome proteins (i.e., peripheral membrane proteins like LRR 2). We have found that if urine is processed immediately, the purified and processed exosome pellets can presumably be stored indefinitely at -80°C for proteomic analysis, and we have verified this out to several weeks. For this and other reasons, including our requirement for at least 70 mL of urine, we cannot use existing banks or frozen samples and thus samples MUST be collected fresh to achieve our aims.

Purification of Exosomes: Several exosome isolation protocols have been compared including ultracentrifugation, ultrafiltration, and immunoprecipitation with magnetic beads. The most reliable method is the ultracentrifugation method, where urine is centrifuged at 500 xG for 10 min, supernatant removed and centrifuged at 17,000 xG for 15 min, and

supernatant removed and finally centrifuged at 150,000 xG for 2 hours to form an exosome pellet. Urine samples that appear cloudy in appearance will be discarded, and exosome preparations associated with samples that demonstrate abnormal urinalysis (abnormal color or appearance, presence of blood cells, bacteria, yeast, or creatinine below 4.8 or above 19 mmol, or protein >20 mg/dL) will be discarded. Exosome samples from patients or controls with known kidney disease and/or abnormal GFR (<60) will also be discarded, to avoid co- variables in exosome presentation due to defective kidney secretion. Wet exosome pellets or exosomes resuspended and sonicated into PBS will be stored at -80°C until desired group sizes for analysis are achieved.

Targeted Proteomic Analysis of late-onset PD proteins. LRRK2 can be efficiently detected via western blot from exosome preparations, but mass spectrometry detection was variable presumably due to low abundance. Because LRRK2 is prioritized, a targeted approach specifically for detection of LR K2 is used. Exosome pellets are first resuspended in PBS, sonicated, and protein quantified by BCA analysis. 50 μg of exosomes are set aside and frozen at -80°C for whole-proteomic analysis. 4 μg of exosome proteins are combined into Ix Laemmli buffer with 5% BME and heated at 70°C for 10 min prior to loading samples onto a 10% TBX 12 well gel (Bio-rad). Samples are electrophoresed and transferred overnight onto PVDF membranes. Successful transfer is verified by Ponceau S incubation of PVDF membranes, and the PVDF is cut into three strips based on molecular weight, where the first strip spans -100 kDa to -400 kDa (LRRK2), the second strip is TGS101 exosome loading control (-25 kDa to -100 kDa). Western blots are repeated in triplicate. Monoclonal antibodies have been discovered and validated to these three targets (UDD-03 for LRRK2, Epitomics, and TGS101, Santa Cruz) and each antibody detects only a single robust band of the appropriate size in fully evaluated immunoblots. The LRRK2 antibody has been validated in both rat and mouse KO tissue, and in human brain samples (single band) and human cell lines. Ratios for signals generated by LRRK2 compared to TGS101 will be determined by chemiluminesence on an Alpha-Innotech Fluorchem HD machine. This approach has been found to have a greater linear range of protein detection than comparable technology by Li -Cor using fluorescence. Ratios for most experiments will be normalized between gels using a reference exosome sample that is arbitrarily defined as 1 ,0, thus multiple western blots can be integrated together with full detection of technical error for each sample.

Unbiased Whole-Proteomic Analysis. 50 g of sonicated exosomes in PBS are supplemented to 50% TFE (trifluoroethanol) and reduced by addition of TCEP (tris(2- carboxyethyl)phosphine) and incubated for 45 min at room temperature, then alkylated by adding iodoacetimide, followed by digestion with trypsin gold (Promega). Tandem mass tag (TMT) labeling reagents (Thermo-Pierce) are added to the samples, incubated, and then supplemented with 5% hydroxylamine to quench reactions. Isobaric labeled peptides from each sample are combined, purified, and loaded onto a custom packed tandem long-flow SCX/C18 column, and MuDPIT analysis is accomplished using a nanp-HPLC (nLC) with a 14 fraction salt bump gradient carried out in duplicate. Peptides are eluted from the analytical column after each salt bump with optimized 105-min reverse-phase solvent gradient. The peptides are directly electrosprayed into a LTQ Orbitrap Velos mass spectrometer operated in a data-dependent mode with dynamic exclusion to enable identification of less abundant ions.

Tandem mass spectra are analyzed using a suite of custom developed bioinformatics tools. The combination of XCorr and DeltaCN is used initially for score optimization, and results are filtered to a 2% peptide false discovery rate (FDR) using concatenated (forward and reverse db's) with the requirement of a minimum of two quantifiable peptides per reported protein.

An exemplary but non-limiting method according to the invention is set forth as follows.

1. Establishing initial urinary exosome samples and pools from cases and controls, and estimate heterogeneity and power in proteomic analysis

The technology for whole proteomic exosome screening (see Figure 4) and LRRK2 measurements (see Figure 2) in control urine samples has been developed. These technologies need to be translated to clinical populations before biomarker searches can move forward in an informed manner. First, intra-individual reproducibility in proteomic measures is established by analyzing the same individuals seen in the Movement Disorder Clinic (disease affected and unaffected) repeatedly over time. Second, 100 PD cases and 100 age and gender matched control samples are collected, using part of the exosome preparations to create a reference pool to be used for run-normalization, and store the remainder of the exosomes for analysis. Third, heterogeneity and power in an initial dataset including 30 cases and 30 controls by whole-proteomic and targeted proteomic analysis is determined, and ultimately the determination of appropriate sample-sizes and replications needed for reasonable power for targets of interest.

First, urine samples are collected during morning clinic hours, assigned random barcode identifiers, and processed for exosome purification on the same day. Clinical phenotype data will be entered into the study database. Exosome pellets are glass-like in appearance (similar to isopropanol R A pellets), and exosome pellets that are discolored or otherwise unusual in visual composition will be noted and marked for possible study exclusion. To provide study uniformity, wet exosome pellets will be resuspended and sonicated in PBS and stored at a standard concentration of 0,5 mg/mL, as determined by microtiter BCA assays, and samples maintained at -80°C. Samples that do not meet study criteria of normal urinalysis and kidney function will be discarded once those test results are made available in the study database.

It is important to establish measure reproducibility over time. Correlation is anticipated between exo somes preparations obtained from the same individual at multiple time points. However, we have not found published evidence that this aspect of urinary biomarkers has been seriously considered. We will derive exosomes from 6 control individuals and 6 PD-affected individuals, derive additional sample from the same individuals 1 week later on a follow-up appointment, and 3 months later for a final appointment. Whole proteomic screening will be performed in addition to targeted assessment of LR K2, The same individual will be analyzed completely in single runs, thus obviating an exosome-pool reference. We will perform a rank-test non-parametric correlation analysis and non-linear regression analysis to establish whether initial protein level estimations significantly predict future estimations. We will interpret a p value of >0.05 as significant correlation.

Second, urine samples are collected as above, and 50 early stage PD cases (Hoehn and

Yahr stage 1-2) and 50 later-stage PD cases (Hahn and Yahr stage 3-5) will be collected, with matching of age and gender distribution between cases and controls. Gender distribution will match that of clinical distribution, roughly 2:1 male to female. Exosome fractions will be produced and 20 g of exosomes from each case and control will be combined together to create a standard exosome pool consisting of ~4 mg of exosomes. This exosome pool will provide an ample supply of protein to normalize between both targeted and unbiased runs as described herein.

Third, after completion, we will use the reference pool to perform an initial statistical analysis of clinical samples to determine our power and to measure statistically significant changes between clinical phenotypes and particular protein targets of interest. For targeted analysis of LRRK2, the described immunoblot approach will be used to quantify LR K2 relative to the ubiquitous exosome marker TSG101, and ratios normalized to the pooled exosome standard. For whole-proteome analysis, groups sizes of 5 (plus the exosome pool control) will be analyzed in individual multi-plex MudPIT runs (x6 isobaric labels are included in the TMT labeling approach), and we have previously established our power to detect at least 935 proteins with high confidence in this multi-plex technique. In this aim, 12 separate 2D nLC-ESI-MS/MS (i.e., MudPIT) analyses will be carried out in duplicate, encompassing 30 PD-affected and 30 control cases, roughly one -third of the samples collected. Global spectral intensity will be normalized within the run, while the standard exosome pool fraction will be used to normalize spectral intensity across runs. The use of a pooled sample to normalize across runs has been widely adopted in efficacious biomarker approaches and greatly reduces false-discovery rates and other common errors. The clinical samples are not pooled for analysis; rather, the pool serves as the best possible reference standard across runs.

2. Determining whether there are exosome proteins significantly associated with PD progression or susceptibility, and whether LRRK2 expression or phosphorylation levels are changed due to Sunitinib (a potent LRRK2 kinase inhibitor) exposure.

Exosome structures have never been formally analyzed in PD, yet these molecules contain many of the proteins linked to PD pathology and genetics. First, age and gender matched samples are continued to be collected from early-stage PD (Hoehn and Yahr 1-2) and late-stage PD (Hoehn and Yahr 3-5) together with control patients. Second, exosome samples will be derived from patients treated with sunitinib, together with control patients, from the Comprehensive Cancer Center. Third, exosome preparations will be analyzed by whole-proteomic screening and targeted protein detection. Fourth clinical phenotypes are correlated and effect size determined for targets of interest for PD progression, susceptibility, and sunitinib treatment.

First, samples from early stage PD (H&Y 1-2), later stage PD(H&Y 3-5), and matched control individuals, are derived as described above, for both targeted and unbiased proteomic analysis. Based on our previous experiences with biomarker studies in clinical samples, we anticipate the collection of another 100 PD-affected and 100 control individuals, to complement the cohort above. However, the sample number based on the above analysis is adjusted to maximize the possible study power, for example by increasing sample replicate runs and decreasing new sample recruitment. The results of power calculations are prioritized, particularly for proteins of interest such as LRRK2 in the exosome samples.

Second, the discovery that LRRK2 is highly expressed in the periphery and kidneys and that the kinase inhibitor sunitinib inhibits LRRK2 with a very impressive potency allows detection of putative pharmacodynamic markers for LRRK2 activity. In experiments in rats, the administration of clinically relevant doses of sunitinib intraperitoneal ly destabilized total LRRK2 protein expression (Figure 3). Kinase-inactivated LRRK2 is also unstable in vivo and in vitro. To collect urine from patients undergoing sunitinib treatment, we will obtain samples of urine four hours post sunitinib dosage and immediately process exosome samples. Both LRRK2 expression is measured and the status of the N-terminal phosphorylation sites is evaluated that have been shown to be responsive (in diminishing) to the inhibition of LRRK2 activity, including p910 and p935, in addition to our developed and sensitive

autophosphorylation antibodies. The error for LRRK2 measurements, as calculated above, helps establish the number of samples that are needed to be collected to detect relevant effect sizes. However, the animal model of rats treated with sunitinib was very compelling in that a large reduction in soluble LRR 2 protein was observed in kidney lysates. In the rat experiments, enough rat urine was not collected to detect LRRK2 because our limit of sensitivity by immunoblot is about 1 pg of LRRK2, so human samples seem required to get the volume needed. After collecting five sunitinib samples and five controls, interim analysis is performed to determine the extent of sunitinib induced differences, and it seems possible that the changes are so robust that no additional samples would be required for significance. Even so, a replication cohort should be generated.

Third, samples collected in the study are processed using unbiased whole-proteomics and targeted measurements of LRRK2 as samples are continued to be collected, using the pooled exosome fraction as a reference sample for cross-run normalization. The pooled exosome fraction serves as the best possible measure of quality control during individual runs, and runs where the pooled standard is not correlated with past runs of the pooled standard will be re-performed.

Fourth, once the complete cohort, likely encompassing ~200 cases and -200 controls, appropriate statistical tests rationally conceived are used to identify clinical pheno types or groups of phenotypes associated with particular exosome proteins or functional classes of exosome proteins.

3. To replicate targets of interest in a new cohort of patients and controls using targeted and unbiased proteomic approaches.

A critical component to biomarker discovery is replication of targets of interest in naive cohorts. Targets of interest identified and prioritized above are thus defined in a new group of patients and controls. In addition, a larger combined dataset can reveal other significant exosome/phenotype associations that can be followed upon in future studies.

EXAMPLE 2

Clinical samples

Urine specimens were collected from the Movement Disorder Clinic at the University of Alabama at Birmingham, and CSF and brain tissue samples were obtained from the NICHD Brain and Tissue Bank at the University of Maryland. Samples were supplemented with 5GmM Tris pH 7.4, ImM AEBSF, 2mM EGTA, 5mM β-Glycerolphosphate, ΙμΜ pepstatin A, and 1 μΜ E-64 before exosome purifications.

Exosome Isolation

Cell supernatants and clinical samples were subjected to a differential centrifugation protocol starting with a 500 x g spin for 10 minutes, a 10k x g spin for 30 minutes, and a 100k x g spin for 2 hours, Pellets were resuspended and centrifuged for a second time at 100k x g in reduced volumes. For further purification in some experiments, exosome pellets were resuspended into ~1 g/ml iodixanol solution and added to the top of an iodixanol gradient followed by centrifugation at 500k x g for 16 hours.

Animals, Cell Culture, and Plasmids

Male WT and LRRK2 KO rats were obtained from Sigma and used at 10-12 weeks of age. Primary neurons and macrophages were prepared from C57BL/6J WT or LRRK2-BAC mice (J AX strain 012467). For primary neuron cultures, NeuN staining was used to confirm cultures that were comprised of >95% NeuN positive cells. For some experiments, LRRK2 was immunoprecipitated using Dynal ProtG Dynabeads together with anti-Myc antibody (Roche) following manufacturer's instructions. HE -293T cells and macrophage Raw264.7 cells were maintained in exosome-free media composed of DMEM with 10% fetal-bovine serum (FBS). Cells were transfected as previously described with WT-LRRK2, G2019S- LRRK2, D1994A kinase dead LRR 2, 14-3-3ε, difopein, scrambled-difopein, or eGFP, in the pcDNA3.1 backbone.

LRRK2 inhibitors and antibodies

The small molecule inhibitor HG-10-102 was synthesized in-house according to the method previously described, and L2inl compound was a gift from Dr. Dario Alessi.

Antibodies to LRRK2 (Epitomics c41-2 and UDD3, and NeuroMab N241 A/34), pS935 (Epitomics UDD2), TSG101 and 14-3-3pan (Abeam), Alix and CD9 (Santa Cruz), tubulin and FLAG (Sigma), myc (Roche), V5 (Invitrogen), and secondary donkey anti-mouse and anti -rabbit conjugated to HRP, Cy2, Cy3, Cy5, and Biotin (Jackson immunoResearch) were used. Antibody ATTO 647N Goat anti-Rabbit (Active Motif) was used for super resolution imaging. Vesicle marker antibodies EEA1 , Rab7,9,l 1 , and AAPL1 were from Cell

Signaling. Lysotracker was from invitrogen.

Immunohistochemistry and Immunofluorescence

Dissociated cells were cultured on glass coverslips and fixed with 4% PFA in PBS for 10 min. at room temperature. Cells were stained for LRRK2 and other targets as previously described. In all experiments, cells from LR K2 KO animals were used to confirm LRRK2 staining. Exosome preparations were resuspended into 0.5% PFA in PBS and incubated for 5 min at room temperature. Glycine was supplemented to 0.1M concentration, and the exosomes pelleted at 500 kG for 15 min. Exosome pellets were resuspended in a blocking buffer (5% exosome-free donkey serum, 0.3% TRITON X-100 in PBS) and incubated for 30 min on a rotating wheel. Primary antibodies were supplemented to 0.01 μg ml, incubated overnight on a rotating wheel, and exosomes pelleted and washed. Secondary antibodies were added at 0.1 μg/ml and incubated for 2 hours. Exosomes were pelleted, washed, resuspended in ProLong Gold (Invitrogen) and plated onto glass coverslips.

Animals were perfused with PBS followed by 4% PFA in PBS, with brain and kidney dissected and allowed to post- fix in 4% PFA overnight. Tissues were embedded with a solution of 30% sucrose for three days, followed by cryo-preservation in isopentane. For brain tissue, coronal sections were cut at 40 μηι on a freezing microtome, with staining procedures performed as previously described. 5 μπι kidney sections were cut from paraffin embedded tissue blocks and dried overnight at 60°C, Sections were deparaffinized and hydrated using ethanol and sections were treated with 0.01 M sodium citrate buffer (pH 6) in a pressure cooker for 5 minutes. Sections were washed and transferred to TBS-T, endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 min, incubated in blocking solutions and antibody solutions as previously described, Diaminobenzidine (DAB; Scy Tek Laboratories) was used as the chromagen and hematoxylin (no. 721 1 , Richard- Allen

Scientific) as the counterstain. LRRK2 KO tissue was used as a control in all experiments. Cryo-EM and Super Resolution Imaging

For cryo-EM, exosomes were resuspended in water, applied to Quantifoil holey film (Quantifoil MicroTools, Jena, Germany) and plunge frozen using an FEI Vitrobot. The samples were observed in an FEI Tecnai F20 200kV field-emission gun microscope equipped with a high-sensitivity Gatan Ultrascan 4000 CCD camera. STED microscopy of

fiuorescently labeled exosomes was performed with a FV300 confocal microscope

(Olympus) and an ultrafast laser system (Chameleon Ultra, Coherent). The STED component was generated by passing part of the laser output through a vortex spiral phase plate (VPP-la, RPC Photonics, Inc.) and the excitation component was filtered out from a supercontinuum generated by focusing the 750nm ultrafast pulses to a 12 cm long photonic crystal fiber (SCG-800, Newport Corp,). Exosome Proteomics

Sonicated exosomes suspended in PBS were supplemented to 50% trifiuoroethanol and reduced by addition of tris(2-carboxyethyl)phosphine and incubated for 45 min at room temperature, then alkylated by adding iodoacetimide, followed by digestion with trypsin gold (Promega). Tandem mass tag (TMT) labeling reagents (Thermo-Pierce) were added to the samples, incubated, and then supplemented with 5% hydroxylamine. Isobaric labeled peptides from each sample were combined, purified, and loaded onto a tandem long-flow SCX C18 column, and MuDPIT analysis was accomplished using a nano-HPLC (nLC) with a 14 fraction salt bump gradient carried out in duplicate. Peptides were eluted from the analytical column after each salt bump with optimized 105-min reverse-phase solvent gradient. The peptides were directly electrosprayed into a LTQ Orbitrap Velos mass spectrometer operated in a data-dependent mode with dynamic exclusion to enable identification of less abundant ions. Resultant spectra were analyzed using a suite of custom developed bioinformatics tools. The combination of XCorr and DeltaCN was used initially for score optimization, with results filtered to a 2% peptide false discovery rate (FDR) using concatenated (forward and reverse db's) with the requirement of a minimum of two quantifiable peptides per reported UniProt object.

Kinase Assays

Recombinant LRRK2 (Δ 1-970) was purchased from Invitrogen and full-length LRRK2 protein was isolated from HEK-293T cells over-expressing LRRK2 protein. Kinase reactions were performed as previously described. Autophosphorylation was monitored using an antibody directed to pi 503 of LRRK2 using the Alpha-Screen kit following manufacturer instructions (Perkin Elmer). Recombinant proteins were assessed for equal purity (>95% by Coomassie SDS-PAGE) and protein concentration as determined by BCA assay (Pierce). In some experiments, kinase activity was determined by coomassie stain and autoradiography film exposure.

Western blotting

Proteins were resuspended into Laemmli buffer and eletrophoresed on either 7.5% or 4-20% gradient TGX gels (BioRad) and proteins transferred onto PVDF membranes. Blue- Native PAGE gels were processed as previously described. Membranes were blocked with 5% milk in TBS-T and antibodies applied per manufacturer instructions. Blots were exposed using Luminata HRP Substrate system (Millipore) and signal intensity recorded using an Alpha-Innotech Fluorchem HD system or autoradiography film. Identification of LRRK2 in Exosomes

LRRK2 action in the endocytic pathway may include aspects of lysosomal and autophagy mediated degradation, receptor recycling, and vesicle budding. Evidence of LRRK2 association with MVBs, the obligate source of exosomes, was observed previously. Our past studies demonstrated that the highest expression of endogenous LRRK2 in mice is in the kidney, and the kidneys are known to secrete high quantities of exosomes into urine. To test whether LRRK2 can be secreted in urinary exosomes, we isolated a 100k x g pellet (PI 00) into lysis buffer together with an equivalent- volume concentrated supernatant from healthy human subjects and measured LRRK2 protein content via western blot (Figure 5A). LRRK2 was present at an approximate concentration of 1 picogram per milliliter of urine, and all of the LRRK2 protein localized to the PI 00 (exosome) fraction. Exosomes are organelles that maintain the orientation of the proteins from the parental cells, so that extracellular proteins or extracellular protein domains remain extra-exosomal, whereas cell cytosolic proteins are encapsulated within the exosome, A trypsin-sensitivity assay demonstrated that the addition of T ITON-X 100 detergent to break the vesicles was required for proteolytic digestion of LRRK2, suggesting that LRRK2 is localized within exosomes (Figure 5B). The LRRK2 binding partner 14-3-3 was also sensitive to trypsin degradation in the presence of detergent. In contrast, CD9 is known to be present in a large trans-lummal protein complex within the plasma membrane that was resistant to trypsin digestion whether detergent was added or not.

Next, the PI 00 fraction was separated on an iodixanol density gradient and LRRK2 was found to float in the same fraction as the canonical exosome proteins Alix and TSG101 (Figure 5C), suggesting that LRRK2 specifically resides within exosomes. CD9 is a transmembrane protein that is abundant in plasma-membrane derived vesicles that traffic to MVBs. We found that CD9 diffusely spreads across the high density fractions, with the highest concentration in the exosome-containing fraction. Under native (non-crosslinked or dehydrated) conditions, the exosomes were perfectly spherical with intra-exosome puncta composing a dense vesicle core, as visualized using cryo-electron microscopy (EM, Figure 5D). We localized LRRK2 immunoreactivity to isolated exosomes using a super-resolution technique (stimulated emission depletion microscopy) and found structures also of -100 nm in size with LRRK2 protein primarily concentrated in the exosome core (Figure 5E). CD 9 has been previously used in fluorescence and immunoaffinity approaches to purify exosomes, but we found complete exclusion of LRRK2 immunoreactivity in CD9 positive exosomes via immunofluorescence in dilute exosome preparations (Figure 5F). In contrast, LRRK2 partially co-localized to TSG101 enriched exosomes, although examples of TSG101 positive exosomes with weak or no detectable LRRK2 expression could also be observed. As CD9 is a ubiquitously expressed plasma membrane protein, this could indicate that the intracellular source of LRRK2-positive exosomes could be distinct from plasma membrane-derived exosomes.

Both LRR 2 dimerization and phosphorylation are activities that have been linked to LRRK2 kinase activity. To assess LRRK2 dimerization, urinary exosomes were resuspended in buffer containing the non-ionic detergent TRITON X-100 and lysates analyzed by native- PAGE (Figure 5G). Urinary exosome LRRK2 migration was identical to that of endogenous and kinase-active LRRK2 isolated from cell cytosols. Low amounts of LRRK2 in preparations without detergent treatment were likely due to exosome breakage during processing, but show that detergent did not alter the migration pattern of LRRK2 in native gels. To measure LRRK2 phosphorylation, 10 μg of total protein from exosomes was compared to 10 μg of total protein isolated from low post- mortem interval human brain cerebral cortex (brain samples described previously for LRRK2 expression), together with 1 ng of recombinant LRRK2 protein derived from HE -293T cells (Figure 5H). The recombinant protein is known to be heavily phosphorylated at serine 935 (pS935). pS935 levels were comparable between recombinant kinase-active LRRK2 and urinary exosome derived LRRK2. In comparison, much lower levels of total and pS935 LRRK2 were detected in human brain.

These results demonstrate that LRRK2 can be readily measured from urinary exosomes and that LRRK2 protein in exosomes derives from the cell cytosol, either captured in vesicles that fuse with MVBs, or packaged during the inward internalization of vesicles fusing with MVBs. Although LRRK2 protein concentration is appreciable in urinary exosomes, the overall levels are not sufficient to directly assess LRRK2 kinase activity with any described kinase activity assay.

Localization of LRRK2 to Kidney Luminal Tubule Cells and Characterization of Urinary Exosome Proteomes

The sources of urinary exosomes are not fully understood, although the kidney is thought to be the major contributor. Staining for LRRK2 in healthy rat kidneys revealed intense LRRK2 expression in virtually all segments of the outer and inner collecting duct epithelium, with reactive cells directly abutting luminal spaces (Figure 6A). In contrast, rat LRRK2 KO kidneys are non-reactive under identical staining conditions, albeit with minor nonspecific reactivity observed in a subset of nuclei (Figure 6B). Since the collecting duct epithelium cells specifically are hypothesized to be contributors towards the urinary exosome pool, these results explain in part the source of LRRK2 in urinary exosomes.

It is possible that LRRK2 protein interactors in exosomes may chaperone and control LRRK2 vesicular association. Although several urinary exosome proteomic studies have been performed, these have been qualitative assessments that have not capitalized on recent and dramatic improvements in proteomic technology. To quantitatively measure the exosome proteome, urinary exosomes were pooled from six healthy controls and the resultant exosomes were split into six equal fractions that were labeled with tandem isobaric mass tags and analyzed by long-column multi -dimensional protein identification (TMT-MudPIT). 5,268 peptides were identified and quantifiable that corresponds to 1 ,673 protein

identifications (Table 1). Of the 1,673 proteins, 965 mapped to unique GeneGO objects with assigned function, and the GO process "cellular component organization" was significantly enriched, owing to the high concentration of components of the actin cytoskeleton and known vesicle components (FDR-adjusted ^S^ lO ^"41 ). A number of proteins associated with neurodegenerative diseases were identified in exosomes besides LRRK2, such as DJ-1 , ApoE and Nerprelysin. LRRK2 interacting proteins including 14-3-3ε (NCBI Accession No.

NP_006752.1), HSP70/90, ezrin/moesin/radixin, and Rab7, were all identified at levels comparable to the most abundant exosome proteins (Figure 6C). Despite previous studies that indicate some of these interactors as potential kinase substrates, we were unable to show that LRRK2 could phosphorylate any proteins from exosome lysates.

Among the urinary exosome proteins detected, several highly expressed proteins were identified that are considered exclusive or highly enriched in organs other than the kidney. These organs include prostate, stomach, pancreas, liver, and colon (Figure 6D), where LRRK2 expression of differing levels can also be detected. Notably, we did not obtain any evidence that brain specific proteins were present in urinary exosomes. Thus, it is possible that LRRK2 protein in urinary exosomes originate from these organs, in addition to the kidney.

14-3-3 binding to LRRK2 controls LRRK2 exosome release

We and others have found that LRRK2 may be tightly bound to heat-shock proteins and 14-3-3 chaperones that may control LRRK2 solubility and oligomerization. We sought to test whether interactions with these proteins may control LRRK2 extracellular secretion. First, we determined that HEK-293T cells transfected with LRRK2 actively secrete exosomes into cell culture media (Figure 7A). While knockdown of all 14-3-3 isoforms in HE -293T cells is difficult to accomplish, a short-peptide inhibitor known as difopein has been developed in HEK-293T cells that effectively acts as a pan 14-3-3 inhibitor by blocking 14-3- 3 dimerization. Transfection of difopein in LRRK2-expressing HEK-293T cells resulted in a very efficient ablation of LRRK2 binding to 14-3-3 proteins, as observed through

iramunoprecipitation assays using a pan-14-3-3 antibody (Figure 7B). In cells expressing both LRRK2 and difopein, LRRK2 could no longer be detected in resultant exosome fractions, yet cytosolic levels of LRRK2 and 14-3-3 (pan) remained unaltered. Likewise, difopein treatment did not have any significant effects on total exosome release, indicating 14-3-3 proteins are dispensable for exosome biogenesis and processing.

Acute LRRK2 kinase inhibition via small molecules causes a reduction in 14-3-3 binding to LRR 2. To test whether acute kinase inhibition mediated loss of 14-3-3 binding would also disrupt LR K2 release in exosomes, we first characterized the two most potent and specific LRRK2 kinase inhibitors described. HG-10-102, that is known to be a selective LRRK2 inhibitor, and the widely utilized L2inl compound, were first defined for potency in a kinase inhibition assay measuring LRRK2 autophosphorylation in vitro (Figure 7C,D). When applied to HEK-293T cells at 1 μΜ concentration, these two compounds had comparable effects in reducing 14-3-3 (pan) binding to LRRK2 and reducing LRRK2 release in exosomes (Figure 7E). Unexpectedly, treatment with L2inl, a known inhibitor of E K5, and possibly Aurora A and CHK2, also blocked overall exosome release in HEK-293T cells, as determined by lower levels of TSGl Ol (Figure 7E) and other markers evaluated such as Alix and CD9. Over-expression of 14-3-3ε, the most abundant 14-3-3 isoform identified in urinary exosomes, restored 14-3-3 binding to LRRK2 and exosome release (Figure 7E).

The cytosolic distribution of LRR 2 may be important for LRRK2 packaging into exosomes. Using immunofluorescence localization in HEK-293T cells over-expressing LRRK2 protein, we observe a diffuse yet punctate cytoplasmic localization of LRRK2

(Figure 8A). in cells co-expressing the small peptide difopein, LRJ K2 redistributes to concentrated perinuclear structures (Figure 8C), whereas exposure to LRRK2 kinase inhibitors renders LRRK2 to skein like structures (Figure 8E,F), consistent with previous reports evaluating L2i l exposures. We found that 14-3-3ε over-expression rescued the normal localization of LRRK2 (Figure 8G,H). These results demonstrate how 14-3-3 binding to LRRK2 alters subcellular localization, where diffuse cytoplasmic distribution correlates to extracellular secretion. Because LRRK2 cytoplasmic distribution appears to critically mediate extracellular secretion, we next co-localized LRRK2 with the exosome marker TSGl Ol in HE -293T cells that constitutively secrete exosomes. While TSGlOl had a discrete vesicular like distribution, LRRK2 was more evenly expressed across the cytosol with rarer examples of discrete vesicle-sized puncta, clearly distinguishing LRRK2 from a canonical vesicular protein (Figure 9 A). Some previous evidence suggests that the G2019S mutation may alter this cytoplasmic distribution to LRJ K2 positive intracellular puncta, so we tested whether this mutation affects LRRK2 secretion. Neither G2019S nor a kinase- inactivating mutation altered overall LRR 2 secretion (Figure 9B). These results are also consistent with past data that these alterations in LRRK2 do not affect binding to 14-3-3 proteins. The other protein linked to autosomal dominant PD, -synuclein, can also be secreted in exosomes. To determine whether LRRK2 may co-exist in the same exosomes as a-synuclein, we transiently co-expressed both proteins in HEK-293T cells, isolated exosomes, and analyzed the composition of the structures by immunofluorescence. No examples of co-localized exosomes were observed, suggesting that α-synuclein and LRRK2 may derive from exosomes of divergent origin. (Figure 9C). a-synuclein is also a binding partner for 14-3-3 isoforms, although the a- synuclein complex in exosomes has not been described.

Quantification of LRRK2 in clinical exosome samples

We collected a cohort of late-onset PD without a family history of disease (n=20) and age-matched control samples (n=T5) from the Movement Disorder Clinic at the University of Alabama at Birmingham, following informed consent and institutional approval, and measured LRRK2 and 14-3 -3 (pan) expression relative to the canonical exosome marker TSG101. Age between the PD and control cohort was similar at 62.8±9.4 and 62.5±13.1, respectively, and the modified Hoehn and Yahr score of the PD group was 2.2±0.38.

We found that both LRRK2 and 14-3-3 (pan) had unexpectedly high variability in both controls and PD, as LRR 2 levels varied in the PD affected group by over two orders of magnitude, in contrast to TSG101 that showed lower variability (Figure 10A). After exclusion of outliers from the series (identified with at least one order of magnitude above or below the population average), there were no significant differences in the expression of LRR 2 or 14-3-3 in PD versus controls (p>0.1, Figure 10B,C), nor did LRR 2 expression correlate well with 14-3-3 (pan) expression (Figure 10D). There were no effects of age or gender on LRRK2 or 14-3-3 expression in urinary exosomes. High levels of LRRK2 in exosomes, more than 50-fold above the cohort average, were detected in one PD-affected subject (Figure 10E). This subject had normal quantities of exosomes in urine and had typical late-onset PD and we did not identify a clinical cause for these extreme levels of LRRK2 (Figure 10F). To determine whether LRRK2 expression in urine changes over time, we collected urine from healthy control subjects over the course of a week at different times of the day and found that LRRK2 levels did not significantly vary within this period of time (Figure 10F shows one such subject, one-way ANOVA p>0.5). These data demonstrate the relative ease of collection and measurement of LRRK2 in clinical samples, but the data emphasize the importance of baseline measures of L RK2 expression due to extreme inter- sample variability,

LRRK2 exosomes secreted by macrophages

As opposed to constitutive exosome secretion in active neurons and HEK-293T cells, other cells, such as macrophages, have been described with strong acute regulation of exosome release. We and others have found that activated macrophages of the periphery and brain express high levels of LRRK2 protein that can be induced with stimulation. Upon lipopolysaccharide (LPS) stimulation of RAW 264.7 macrophage cell line that express endogenous LRRK2 protein, we find that the exosome associated protein TSG101 nearly evacuates from the cell cytosol, and abundant exosomes were released that contained high levels of LRRK2 expression (Figure HA). Thus, past assessments of the magnitude of LRRK2 induction in macrophages would be underestimates if extracellular LR K2 were not measured. To confirm and expand on these results, we isolated into culture primary macrophages from the mouse peritoneum of LRRK2 transgenic mice that express mouse WT-LRRK2 or G2019S-LRRK2 from BAC transgenes. Stimulation of these cells with LPS resulted in abundant LRR 2 secretion, but again the G2019S~mutation had no effect on the total quantity of LRRK2 released (Figure 11B,C).

Due to the high levels of LRR 2 expressed in primary macrophages,

immunofluorescence becomes possible. While LPS had a striking effect on overall macrophage morphology from a polarized cell into an amoeboid body, a more modest perinuclear accumulation of LRR 2 and the remaining TSG101 could be observed (Figure 11D). We did not notice an enhanced co-localization between LRR 2 and after stimulation, suggesting that LRRK2 vesicular association is probably independent of stimulation, but overt release is critically modified by stimulation. Macrophage-derived exosomes provide an example of how LRRK2 exosome release can be physiologically regulated.

LRRK2 exosomes in CSF and secretion by neurons

In the brain, serial sectioning confirmed that the most intense LRJ J 2 staining was in the striatum, and many of the LRRK2 positive cells were grouped into patches resembling striosomes juxtaposed against the lateral ventricles (Figure 12A). We rationalized that LRR 2 may be secreted into the CSF from these neurons and other LRRK2 positive cells in the brain. Two samples of CSF were prepared from control subjects and exosomes verified using cryo-EM (Figure 12B). LRR 2 expression was determined at a concentration of -10 picograms per milliliter of CSF in these samples (Figure 12C). To test whether pathogenic LR K2 mutations affect neuronal LRRK2 exosome secretion, viral transduction of cultured neurons resulted in high levels of LRRK2 in exosomes, but these levels did not vary due to the presence of pathogenic mutations (Figure 12D,E). Within neurons, LRRK2 occasionally localized with TSG101 positive vesicles, particularly in dendrites (Figure 12F). As neurons have been previously described as major sources of exosomes in the brain, it seems likely that the source of LRR 2 positive exosomes in the CSF derives from neurons expressing

LRRK2.

These results indicate a pathway for LRRK2 secretion in exosomes (Figure 13).

Exosomes may be recruited to VBs through plasma membrane derived vesicles enriched in transmembrane plasma proteins such as CD9 in a clathrin-dependent manner, or through the direct uptake of late-endosomal vesicles into MVBs. LR K2 cytoplasmic association with late-endosome vesicles is dependent on 14-3-3 binding. As opposed to canonical vesicular proteins, 14-3-3 bound LRRK2 is relatively diffuse and soluble, and this is critical for packaging into MVBs. During the process of vesicle uptake into MVBs, L RK2 is likely captured inward into invaginating late-endosomal vesicles in the cytosol (Figure 11). Cells can then release LRRK2-containing exosomes after MVB docking to the plasma membrane in either a tightly controlled manner, such as in macrophages, or a constitutive manner, such as in HEK.-293T cells, or ceils may traffic LRRK2-positive MVBs to other parts of the cell, such as along dendrites of a neuron, for possible release at a physical proximity different than the originating vesicle, and to a cell that does not normally express LRR 2 protein.

EXAMPLE 3

The present invention provides protocols optimized for purification of LRRK2 protein from a sample (e.g., urine and/or CSF) for utilization as a biomarker for disease state and phenotype. Whereas LRRK2 would be otherwise undetectable in standard clinical protocols to measure protein in urine, with the combination of rapid thawing of clinical samples with vortexing, optimized differential centrifugation that removes extraneous protein that would otherwise inhibit detection of LRR 2 in protein assays, optimized lysis of exosome structures that requires both high concentrations of denaturing detergents as well as sonication optimization, and western blot technology using optimized conditions with specific antibodies sensitive to low picogram to ferntogram levels of protein, the present invention provides a protocol for measuring LRRK2 in a urine and/or CSF sample. Routine handling of urine without rapid freeze thaw will destroy exosomes that harbor LRRK2, standard centrifugation approaches would not be sufficient to isolate LRRK2 fractions, standard lysis conditions would inefficiently lyse exosomes and LRRK2 containing exosomes to free the protein for detection, and standard protein detection assays would not be sensitive enough to detect LRRK2 at the low picogram to high femtogram levels of this invention due to problems with electrophoresis, protein transfer, and antibody-based detection methods.

Thus in the present invention, for the detection of LRRK2, steps that are not routine and conventional include:

-Rapid thawing of unprocessed (raw) urine or CSF at about 37°C to about 55°C (e.g., 42°C) in about 5 ml to about 40 ml aliquots, in polypropelene tubes or other shatter- resistant vessels, for about 1 to 5 min at about 50 to 150 RPM in a shaking water bath. -Samples are processed using differential ultracentrifugation (e.g., multiple spins at increasing G forces) with final centrifugation at speeds of about 500 kG in thick wall tubes optimized for isolation of LRRK2 containing exosomes.

-Lysis into a buffer anchored by about 1% to about 4% SDS. Even with very high SDS, LRRK2 exosomes still require sonication energy to fully lyse and be available for detection.

-Non routine western blotting conditions, which include the use of acrylamide SDS PAGE gels (e.g., commercial pre-cast gels such as 7.5% TGX, Bio-Rad), optimized transfer to nitrocellulose (e.g.,PVDF) membranes, with about 4 hour to about 24 hour transfers (e.g., about!6 hour transfers) in a glycine/10% to 20% methanol buffer, and blocking conditions and antibodies optimized for this assay (e.g., monoclonal antibody C41-2, monoclonal antibody N241/34, monoclonal antibody UDD-03 and/or an antibody to P1292). Incubation times of assays are also optimized for the highest signal to noise ratios.

Although selected embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. TABLE 1. Esosoma! proteins determined by TMT-MudPIT

Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID ikDa Probability Guidelines) Intensity stdev stdev

Oviduct-specific

glycoprotein I OVGP1 75.36 1 .00 3 21.00 3.57 17.02

Ezrin EZRI 69.35 1.00 11 20.00 1.78 8.92

14-3-3 protein zeta/delta 1433Z 27.71 1.00 4 20.00 2.44 12.22

H3BV9

Anamorsin 0 26.41 1.00 2 20.00 3.69 18.43

Dipeptidase 1 DPEPl 45.63 1.00 5 20.00 1.75 8.74

Ras-related protein Rab-8B RAB8B 23.55 1.00 2 20.00 1.65 8.25

Podocalyxin PODXL 58.58 1.00 4 20.00 1.98 9.92

B5BU3

Annexin 8 38.64 1.00 3 19.00 1.75 9.21

Glutamyl aminopeptidase AMPE 109.36 1.00 11 19.00 1.99 10,45

Vacuolar protein sorting- associated protein 4B VPS4B 49.25 1.00 6 19.00 2.71 14.25

Alpha-enoiase ENOA 47, 12 1.00 3 18.00 1.92 10.64

E7ENQ

Annexin 5 30.83 1.00 3 18.00 2.33 12.94

Glutathione S-transferase

Al GSTA1 25.60 1.00 5 18.00 1.76 9.78

Heat shock 70 kDa protein

1A/1B HSP71 69.99 1.00 8 18.00 3.04 16.90

Fructos e- b i sphosphate

aldolase B ALDOB 39.43 0.99 2 18.00 2.02 1 1 ,23

Low-density lipoprotein

receptor-related protein IB LRP1B 515.14 1.00 10 17,00 2.16 12.72

Syntenin-1 SDCB1 32.41 1.00 5 17.00 1.52 8.92

Annexin A2 ANXA2 38.56 1.00 6 17.00 3.28 19.30

Serine/threonine-protein

kinase WNK3 WNK3 198.27 0.99 3 17.00 2.64 15.55

Epidermal growth factor

receptor kinase substrate 8- like protein 2 ES8L2 80.55 1.00 6 16.00 2.48 15.48

Keratin, type 0 cytoskeletal

4 K2C4 57.23 1.00 9 16.00 4.01 25.04

Keratin, type II cytoskeletal

5 K2C5 62.32 1.00 5 16.00 1.88 1 1.72

Protein FAM184B F184B 120.95 1.00 6 16.00 3.14 19.63

Vacuolar protein sorting- associated protein 28

homolog VPS28 25.39 1.00 2 16.00 2,40 15.03

Choline transporter-like E7ETY

protein 2 3 80.14 1.00 2 16.00 3.91 24.47

Calpain-7 CAN7 92.58 1.00 4 16.00 2.56 16.02

Regulating synaptic

membrane exocytosis

protein 2 RIMS2 160.29 1.00 2 16.00 1.1 1 6.94

G-prote in-coupled receptor

family C group 5 member

C Q9NXI0 17.81 1 ,00 4 15.00 2,89 19.23 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID ikDa Probability Guidelines) Intensity stdev stdev

Neurotrophin receptor- interacting factor homolog ZN274 74.1 1 1.00 3 15.00 2.83 18.88

Brain-specific angiogenesis

inhibitor 1 -associated

protein 2 BAIP2 60.81 1.00 7 15.00 2.03 13.53

Vacuolar protein sorting- associated protein 4A VPS4A 48.85 1.00 7 15.00 1.97 13.12

14-3-3 protein gamma 1433G 28.27 0.88 3 15.00 1.93 12.89

Thrombospondin type-1

domain-containing protein

7A THS7A 185.22 0.99 3 15.00 1.35 9.00

Neutrophil defensin 1 DEFI 10.18 1.00 2 14.00 1.89 13.50

Alpha-protein kinase 1 F5H138 129.85 1.00 3 14.00 3.04 21.69

Fructose-bisph osphate

aldolase A ALDOA 39.38 1.00 4 14.00 1.29 9.25

Prostatic acid phosphatase PPAP 44.52 LOO 3 14.00 3.21 22.93

Argininosuccinate synthase 46.48 1.00 5 14.00 1.01 7.18

STAT3- interacting protein

as a repressor SIPAR 27.99 0.98 2 14.00 1.79 12.81

Tripeptidyl-peptidase 1 TPP1 61.19 1.00 2 14.00 2.57 18.39

PHD finger protein 7 PHF7 43.72 1.00 3 14.00 2.24 16.01

Programmed cell death

protein 6 PDCD6 21.84 1.00 3 14.00 3.05 21.78

Beta/gamma crystalSin

domain-containing protein

3 CRBG3 1 16.1 1 0.99 4 13.00 2.53 19.47

Pro-epidermal growth

factor EGF 133.89 1.00 6 13.00 1.35 10.37

Protocadherin-17 PCD 17 126.13 1.00 2 12.00 2.23 18.56

Ryanodine receptor 1 RYR1 564.80 0.99 2 12.00 2.00 16.67

Fructose-b isphosphate

aldolase C ALDOC 39.41 0.94 4 1 1.00 0.77 7.03

E9PDK

Annexin 5 45.68 LOO 8 1 1.00 1.73 15.71

C-C motif chemokine 20 CCL20 10.74 0.99 2 1 1.00 1.04 9.44

Glutathione S-transferase P GSTP1 23.32 1.00 2 1 1.00 1.47 13.37

Phosphatase and actin

regulator 2 PHAR2 69.64 LOO 5 1 1.00 1.43 13.03

Abnormal spindle-like

microcephaly-associated

protein ASPM 409.52 1.00 13 1 1.00 1.59 14.47

Chromodomain-hel icase-

DNA-binding protein 2 CHD2 21 1.20 0.98 4 10.00 1.47 14.74

Rho GTPase-activating

protein 29 RHG29 141.96 0.98 2 10.00 1.77 17.70

Vacuolar protein sorting- associated protein 13A VP13A 360.03 LOO 4 10.00 2.29 22.86

1-phosphatidy linos itol 4,5- bisphosphate

phosphodiesterase eta- 1 PLCH1 189.09 0.99 4 10.00 1.46 14.62 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa) Probability Guidelines) Intensity stdev stdev

CLK4-associating F8WAG

serine/arginine-rich protein 9 70.23 1.00 4 10.00 1.1 1 1 1.1 1

Heat shock cognate 71 kDa

protein HSP7C 70.84 0.91 7 10.00 1.82 18.16

Heat shock-related 70 kDa

protein 2 HSP72 69.96 0.90 7 10.00 1.82 18.16

Proprotein convertase

subtilisin/kexin type 9 PCSK9 74.22 1.00 2 10.00 0.98 9.83

Tigger transposable

element-derived protein 7 TIGD7 63.18 0.91 2 10.00 1.1 1 1 1.12

Q7Z4X

M025-like protein 0 39.73 0.99 3 10.00 1.45 14.52

TBC1 domain family

member 8 TBCD8 130.73 0.98 3 10.00 3.53 35.26

Chromodomain-helicase- DNA-binding protein 1 CHD1 196.55 0.75 2 10.00 0.93 9.33

Dynein heavy chain 1 ,

axonemal DYH14 399.62 LOO 9 10.00 1.56 15.58

Keratin, type 0 cytoskeletal

79 K2C79 57.78 0.93 4 10.00 1.30 12.95

Phosphatidyiethanolamine- binding protein 1 PEBP1 21.03 1.00 3 10.00 2.03 20.31

Ubiquitin-conj ugating

enzyme E2 variant 3 UEVLD 52.21 1.00 3 10.00 2.08 20.82

Ang iotensin-converting

enzyme ACE 149.60 1.00 3 9.00 2.44 27.06

Peptidyl-prolyl cis-trans

isomerase A Q567Q0 1 1.39 1.00 2 9.00 1.60 17.77

Integrin alpha- 3 ITA3 116.52 1.00 2 9.00 1.58 17.55

2',3'-cyclic-nucleotide 3'- phos ph od iesterase CN37 47.53 1.00 2 9.00 0.83 9.20

Ankyrin-3 ANK3 480.10 1.00 7 9.00 1.77 19.71

B7ZM8

C4orf8 protein 5 138.20 1.00 4 9.00 1.26 14.01

EH domain-containing

protein 4 EHD4 61.12 1.00 4 9.00 1.98 21.98

Fibrinogen beta chain FIBB 55.87 1.00 2 9.00 1.14 12.72

Napsin-A NAPS A 45.34 1.00 3 9.00 0.85 9.49

Zinc finger protein 521 ZN521 147.75 0.77 2 9.00 1.62 18.01

Kanadaptin NADAP 88.74 0.98 2 9.00 1.43 15.89

Protein DJ-1 PA K7 19.86 1.00 2 9.00 1.29 14.31

VPS 10 domain-containing

receptor SorCS2 SORC2 128.05 0.99 4 9.00 1.74 19.28

Armadillo repeat- containing X-l inked protein

5 ARMX5 62.29 0.98 2 8.00 2.72 34.03

Helicase-like transcription

factor HLTF 1 13.84 1.00 5 8.00 1.51 18,82

Solute carrier family 12

member 3 S 12A3 1 13.05 1.00 2 8.00 1.08 13.52 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID fkDa) Probability Guidelines) intensity stdev stdev

Disks large homolog 2 DLG2 97.47 1.00 2 8.00 1.50 18.77

Zinc finger protein 326 ZN326 65.60 0.95 2 8.00 1.31 16.40

Histone-lysine N- methyltransferase SETD2 SETD2 287.40 1.00 5 8.00 1.04 12.95

Ig alpha- 1 chain C region IGHA1 37.61 1.00 3 8.00 1.10 13.72

Kinesin-like protein KIF9 KIF9 89.91 0.97 2 8.00 1.25 15.63

Mannan-binding lectin

serine protease 1 MASP1 79.18 0.99 2 8.00 1.65 20.66

Nebulin NEBU 772.42 LOO 13 8.00 0.89 1 1.19

R o-related GTP-binding

protein RhoC RHOC 21.97 1.00 2 8.00 1.21 15.11

Zinc finger protein 423 Z 423 144.49 0.82 2 8.00 1.51 1 8.81

Probable leucine—tRNA

ligase, mitochondrial SYLM 101.89 0.99 2 8,00 0.68 8.54

Probable R A-binding

protein 19 RBM19 107.25 LOO 4 8.00 1.18 14.76

Protein PRRC2B PRC2B 242.80 LOO 6 8.00 1.16 14.53

HEAT repeat-containing

protein 8 HEATS \ ^+ 5. 0.98 4 7.00 1.34 19.08

Keratin, type I cytoskeleial

20 K1C20 48.44 0.95 4 7.00 1.06 15.10

Muscarinic acetylcholine

receptor Ml ACM1 51.37 0.98 3 7.00 1.85 26.49

UropIakin-2 UPK2 19.41 LOO 3 7.00 0.93 13.33

ANKRD26 protein A1L497 198.07 LOO 5 7.00 1.88 26.91

NACHT and WD repeat

domain-containing protein

I NWD1 174.42 0.92 3 7.00 2.02 28.92

Nucleolar GTP-binding

protein 2 NOG2 83.59 0.99 4 7.00 1.04 14.78

Prostaglandin reductase 1 PTGR1 35.83 0.99 2 7.00 0.77 11.06

Bladder cancer related CD9 Q56CY

variant 1 20.72 1.00 2 7.00 1.32 18.80

General receptor for

phosphoinositides 1~

associated scaffold protein GRASP 42.58 0.99 5 7.00 0.74 10.57

Lysophosphatidic acid

receptor 2 LPAR2 39.04 LOO 3 7.00 1.48 21.15

Peptidyl-tRNA hydrolase

ICT1 , mitochondrial ICT1 23.60 0.77 2 7.00 1.12 15.96

Schlafen family member 14 SLN14 103.82 LOO 2 7.00 0.92 13.17

Shugoshin-like 2 SGOL2 144.63 LOO 4 7.00 2.00 28.63

SNRPB protein Q15182 29.63 LOO 4 7.00 0.83 1 1.80

Spectrin repeat containing, 1004.5

nuclear envelope I Q5JV23 4 0.97 7 7.00 1.35 39.34

Spectrin-like protein of the Q7RTM

nuclear envelope and Golgi 4 965.72 0.97 7 7.00 1.35 19.34

TPR and ankyrin repeat- containing protein 1 TRNK1 335.99 LOO 7 7.00 1.18 16.89 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID ikDa) Probability Guidelines) Intensity stdev stdev

Lys osomal -trafficking

regulator LYST 428.85 1.00 3 7.00 1.13 16.1 1

Pendrin S26A4 85.65 1.00 2 7.00 2.23 31 ,88

Serum albumin ALBU 69.30 1.00 9 7.00 1.06 15.20

Zinc finger protein 709 ZN709 74.58 1.00 5 7.00 0.90 12.84

Zinc finger protein 273 ZN273 64.91 0.97 3 7.00 1.40 20.05

Vitronectin VTNC 54.25 0.99 3 7.00 0.68 9.67

Glutathione synthetase GSHB 52.33 0.96 2 6.00 1.10 18.25

Histone acetyltransferase

p300 EP300 263.97 1.00 8 6.00 0.71 1 1 .84

IgGFc-binding protein FCGBP 571 .62 1.00 4 6.00 0.89 14.82

Caspase-2 CASP2 50.63 1.00 3 6.00 0.63 10.46

Death- inducer obliterator 1 DIDOl 243.71 1.00 5 6.00 2.16 36.01

Ras-related C3 botulinum

toxin substrate 1 RACI 21.42 1.00 3 6.00 1.07 17.86

Ankyrin repeat domain- containing protein 12 ANR12 235.49 1.00 10 6.00 2.10 34.97

Breast cancer type 2

susceptibility protein BRCA2 383.97 1.00 7 6.00 0.97 16.24

CAP-Gly domain- containing linker protein 1 CLIP I 162.13 1.00 4 6.00 0.76 12.70

Charged multivesicular

body protein 5 CHMP5 24.54 0.99 ₂ 6.00 1.24 20.71

Diffuse panbronchiolitis

critical region protein 1 DPCR1 56.29 0.93 2 6.00 0.19 3.13

Disintegrin and

metalloproteinase domain- containing protein 29 ADA29 92.68 0.98 3 6.00 1.23 20.45

DNA repair protein

complementing XP-G cells ERCC5 133.01 0.94 3 6.00 0.82 13.67

Leucine-rich repeat- containing protein 8A LRC8A 94.12 0.91 2 6.00 0.81 13.57

Microtubu 1 e-associated

protein 1A MAPI A 305.28 1 .00 4 6.00 0.79 13.21

Mitogen-activated protein

kinase kinase kinase 15 M3K15 147.33 0.99 3 6.00 0.84 13.95

Myosin- 10 MYHI 0 228.84 0.99 4 6.00 1.42 23.65

Paternally-expressed gene 3

protein PEG3 180.70 1.00 6 6.00 1.92 32.02

PIN2/TERF 1 -interacting

telomerase inhibitor 1 PINX1 36.99 0.89 2 6.00 1.48 24.63

Protein FAM135A F135A 169.71 1.00 4 6.00 0.89 14.85

Protein FAM193B F193B 96.47 0.86 3 6.00 0.85 14.18

Rab-3 A- interacting protein RAB3I 52.97 1.00 5 6.00 1.36 22.67

Ras-related protein Rab-7a RAB7A 23.46 1.00 3 6.00 0.84 13.94

Solute carrier family 15

member 2 S15A2 81.71 1.00 2 6.00 0.79 13.17

Transcription factor SOX-3 SOX3 45.16 0.96 3 6.00 1.80 29.96 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID OiDa ¹ ) Probability Guidelines) Intensity stdev stdev

Epidermal growth factor

receptor kinase substrate 8- like protein 1 ES8L1 80.18 1.00 2 6.00 1.05 17.52

Galectin-3-binding protein LG3BP 65.27 1.00 3 6.00 1 ,32 22.07

Microtubule-associated

protein IB MAP IB 270.45 1.00 8 6,00 1.09 18.21

SWI/SNF-related matrix- associated actin-dependent

regulator of chromatin

subfamily A containing

DEAD/H box 1 SMRCD 117.31 1.00 5 6.00 0.87 14.49

Tetratricopeptide repeat

protein 18 TTC18 125.59 1.00 3 6.00 1 ,46 24.27

Uveal autoantigen with

coiled-coil domains and

ankyrin repeats UACA 162.39 3 .00 6 6.00 0.72 12.05

Zinc finger protein 714 Z 714 63.97 1.00 3 6.00 1.72 28.71

Aquaporin-2 AQP2 28.80 1.00 3 6.00 1.15 19.20

Vacuolar protein sorting- associated protein VTA1

homo log VTA1 33.84 1.00 2 6.00 0.99 16.51

EH domain-containing

protein 1 EHD1 60.57 1.00 4 6.00 2.14 35.64

CD2-associated protein CD2AP 71.39 1.00 4 5.00 2.18 43.61

Origin recognition complex

subunit 5 ORC5 50.23 1.00 3 5.00 0.76 15.19

Putative aspartate

aminotransferase,

cytoplasmic 2 AATC2 47.26 0.92 2 5.00 0.95 19.05

Vitamin K-dependent

gam ma-carboxyl ase VKGC 87.49 0.99 3 5.00 1.41 28.19

Zinc finger protein 93 ZNF93 70.91 0.94 3 5.00 0.79 15.80

Ficolin-2 FCN2 33.96 1.00 2 5.00 1.36 27.28

Matrix-remode 1 ing- associated protein 8 MXRA8 49.08 1.00 2 5.00 1.16 23.13

Rho GTPase-activating

protein 7 RHG07 170.47 0.97 2 5.00 1.05 21 .06

Voltage-gated sodium

channel alpha subunit

splice variant SCN3A-S Q9C007 221.36 1.00 5 5.00 1.03 20.66

40S ribosomal protein S24 E7EPK6 32.39 1.00 3 5.00 0.62 12.41

Collagen alpha- 1 (XXVIII)

chain COSA1 116.57 0.93 2 5.00 1.24 24.83

Cycl in-dependent kinase 12 CDK12 164.04 1.00 7 5.00 0.40 8.06

Estradiol 17-beta- dehydrogenase 2 DHB2 42.74 0,95 2 5.00 0.95 18.97

Paired amphipathic helix

protein Sin3b SIN3B 132.97 0.98 2 5.00 0.89 17.82

Apolipoprotein E APOE 36.1 1 1.00 2 5.00 1.21 24.15 Total

Protein Total Peptides

Weight Group (Paris % rel

Seauence Name Gene ID OtDa) Probability Guidelines) Intensity stdev stdev

E3 ubiquitin-protein ligase

R F213 RN213 591.01 1.00 10 5.00 1.04 20,86

Formin-2 FMN2 179.98 0.99 4 5.00 0.76 15.13

Hi stone H4-like protein

type G H4G 10.98 3 .00 2 5.00 1.00 19.97

Hyaluronan mediated

motility receptor HMMR 84.03 0.88 3 5.00 0.50 10, 10

Keratin, type II cytoskeletai

1 K2C1 65.98 1.00 4 5.00 1 .13 22.53

MORC family CW-type

zinc finger protein 4 MORC4 106.26 0.99 3 5.00 0,66 13.16

Myosin-3 MYH3 223.75 1.00 6 5.00 0.63 12.57

Myotubularin-related MTMR

protein 1 1 B 79.48 0.77 2 5.00 0,72 14,41

Na(+)/H(+) exchange

regulatory cofactor NHE- RF3 NHRF3 57.08 1.00 2 5.00 0.66 13.26

Neuron navigator 3 NAV3 255.47 0.99 5 5.00 0.87 17.31

Probable E3 ubiquitin- protein ligase C12orf51 K0614 439.05 1.00 7 5.00 1.17 23.44

Rab GTPase-activating

protein 1 RBGP1 121.64 1.00 6 5.00 1.43 28,66

Ras-related protein Rab-

27B RB27B 24.57 0.99 2 5.00 0.71 14.20

RNA exonuclease 1

homolog REXOl 131.41 1.00 7 5.00 0.89 17.79

Sacs in SACS 520.78 1.00 6 5.00 0.94 18.76

Solute carrier family 2,

facilitated glucose

transporter member 5 GTR5 54.92 1.00 2 5.00 1.81 36.28

Sorbin and SH3 domain- containing protein 1 SRBS1 142.41 1.00 6 5.00 1.22 24.36

Sorcin SORCN 21.64 1.00 3 5.00 0.58 1 1.69

Sorting nexin- 18 SNX18 68.83 1.00 3 5.00 0.74 14.89

Splicing factor,

arginine/serine-rich 19 SFR1 139.17 1.00 6 5.00 0,60 11.96

Striated muscle

preferentially expressed

protein kinase SPEG 354.05 1.0Q 8 5.00 0.50 10.04

Transcription initiation

factor TFIID subunit 3 TAF3 103.50 0.96 3 5.00 0,61 12, 12

Xylosyl transferase 1 XYLTI 107.48 1.00 6 5.00 0.69 13.79

Zinc finger protein 559 ZN559 62.26 0.99 2 5.00 1.26 25.30

Zinc finger protein 624 ZN624 99.85 1.00 3 5.00 1.08 21.55

Zinc finger protein 654 ZN654 65.79 1.00 4 5.00 0.85 17.04

Kinesin-like protein KIF23 KIF23 109.97 1.00 3 5.00 1 ,03 20.51

Remodeling and spacing

factor 1 RSF1 163.70 0.98 3 5.00 0.61 12.27

SH3 and PX domain- containing protein 2B SPD2B 101.50 0.98 2 5.00 0.29 5.83 Total

Protein Total Peptides

Weieht Group (Paris % rel

Sequence Name Gene ID fkDa) Probability Guidelines) Intensity stdev stdev

Zinc finger protein 415 ZN415 68.74 0.90 2 5.00 1.17 23.47

OIfactomedin-4 OLFM4 57.23 LOO 6 5.00 1 .66 33.21

Probable ATP-dependent

RNA helicase DDX41 DDX41 69.77 1.00 4 5.00 0.96 19.22

RNA -binding protein 44 RBM44 1 17.89 1.00 4 5.00 1.01 20.15

Chromodomain-helicase- DNA -bin ding protein 6 CHD6 305.20 1.00 6 5.00 0.70 13.94

Q7Z5N

Proline rich 2 protein 0 27.29 0.97 3 5.00 0.94 18.71

Structural maintenance of Q86VX

chromosomes 3 4 141.40 1.00 5 5.00 1.30 25.98

WD repeat-containing

protein 35 WDR35 133.44 0.99 4 5.00 0.93 18.62

ATP-binding cassette A7BKA

protein 4 138.53 0.99 3 5.00 1.18 23.57

Peroxisomal carnitine 0- octanoyltransferase OCTC 70.12 0.99 2 4.00 0.46 11.39

Transmembrane channellike protein 7 TMC7 83.43 0.93 2 4.00 0.62 15.59

Histone- lysine N- methyltransferase MLL2 MLL2 593.00 1.00 9 4.00 0.74 18.56

AF4/FMR2 family member

4 AFF4 127.36 0.98 4 4.00 0.93 23.17

CASP8AP2 protein A7E2C0 222.36 1.00 9 4.00 0.43 10.85

Cell cycle progression A8K9T

protein 1 0 93.41 1.00 5 4.00 0.80 19.93

Dynein heavy chain 8,

axonemal DYH8 514.32 0.97 6 4.00 0.84 21.01

Eosinophil peroxidase PERE 80.97 0.95 2 4.00 0.41 10.28

F-box only protein 5 FBX5 50.10 0.89 2 4.00 0.86 21.48

Glycogen phosphorylase,

muscle form PYGM 97.01 0.99 2 4.00 1.14 28.54

Mitotic spindle assembly

checkpoint protein MAD1 MD1L1 83.00 0.99 2 4.00 1 .09 27.16

Nuclear pore membrane

glycoprotein 210-like P210L 210.45 1.00 4 4.00 1.06 26.39

Protein phosphatase 1

regulatory subunit 12A MYPT1 1 15.19 0.93 3 4.00 0.72 17.91

Protocadherin Fat 2 FAT2 479.00 0.99 5 4.00 1.07 26.65

Ras-related protein Ral-A RALA 23.53 1.00 4 4.00 0.43 10.69

Zinc finger protein 182 ZN182 73.58 0.98 2 4.00 0.73 18.22

Cadherin-related family

member 3 F5H0Z2 97.97 0.97 2 4.00 0.61 15.28

Chromodomain -hel icase- DNA-binding protein 3 E9PG89 232.87 LOO 7 4.00 1.48 37.05

Cohesin subunit SA-2 STAG2 141.22 0.86 2 4.00 1.11 27.66

Macrophage-capping

protein CAPG 38.46 LOO 2 4.00 1.04 25.99

MAP7 domain-containing

protein 3 MA7D3 98.35 1.00 4 4.00 0.45 1 1.37 Total

Protein Total Peptides

Weight Group (Paris % rel

Se iuence Name Gene ID OiDa) Probability Guidelines) Intensity stdev stdev

Poly [ADP-ribose]

polymerase 14 PARI 4 202.65 1.00 4 4.00 0.73 3 8.19

Voltage-dependent L-type

calcium channel subunit

alpha- 1 C CAC1 C 248.80 0.97 5 4.00 0.88 21 .97

Zinc finger protein 663 ZN663 12.41 0.91 2 4.00 1.07 26,80

HOYNU

Bloom syndrome protein 5 144.36 1.00 3 4.00 0.88 21 ,99

Breast carcinoma amplified AOAVG

sequence 1 61.63 1.00 4 4.00 0.86 21.52

Elongation factor 1 -alpha 2 EF1A2 50,42 0.96 2 4.00 1.74 43.59

1 -phosphatidylinositol 4,5- bisphosphate

phosphodiesterase beta-2 PLCB2 133.92 0.94 4 4.00 0.96 23.92

Actin filament-associated

protein 1 AFAP1 80.66 1.00 3 4,00 1.38 34.47

Autophagy-related protein

2 homolog A ATG2A 212.71 0.98 2 4.00 1.41 35,21

Basement membrane- specific heparan sulfate

proteoglycan core protein PGBM 468,51 1.00 3 4.00 1.36 34,02

Cation-independent

mannose-6-phosphate

receptor MPRI 274.18 1.00 5 4.00 0.42 10.40

Centromere protein F CENPF 367.52 1.00 5 4.00 1.23 30.71

Delta- like protein 4 DLL4 74.54 0.79 2 4.00 0.67 16.64

Disks large homolog 1 DLG1 100.38 0.91 2 4.00 0.68 17,06

Disks large homolog 5 DLG5 213.72 0.98 3 4.00 0.35 8.64

DNA-directed RNA

polymerase, mitochondrial RPOM 138.51 1.00 8 4.00 1.09 27.33

E3 SUMO-protein ligase

RanBP2 RBP2 357.96 1 .00 4 4.00 1.00 24.94

E3 ubiquitin-protein ligase

UBR2 UBR2 200.39 0.99 6 4.00 0.67 16.64

Envoplakin EVPL 231.44 1.00 3 4.00 0,68 ' 16.98

Fanconi anemia group I

protein FANCI 149.21 1.00 4 4.00 0.95 23.66

Q9HBB

HC56 9 118.66 1 ,00 4 4.00 0.33 8.32

Heat shock 70 kDa protein

6 HSP76 70.97 0.99 7 4.00 1.17 29.13

Hemicentin-1 HMCN1 612,98 1.00 10 4.00 0.60 14.97

Histone-Iysine N- methyltransferase MLL5 MLL5 204.82 0.99 6 4.00 0.42 10.41

Insulin-like growth factor 1

receptor IGF1 R 154.68 0.98 4 4.00 0.56 14,07

Kinesin-like protein

IF13B KU 3B 202.52 0.95 3 4.00 1 .33 33.33

Kinesin-like protein KIF7 IF7 150.48 1.00 2 4.00 0.87 21.64 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa) Probability Guidelines) Intensity stdev stdev

L-lactate dehydrogenase B

chain LDHB 36.60 1.00 5 4.00 0.89 22.28

Myosin-1 1 MYH1 1 227.18 1.00 6 4.00 0.86 21.39

Palmitoyltransferase

ZDHHC3 ZDHC3 37.29 0.79 2 4.00 1.02 25.61

Periplakin PEPL 204.61 1.00 6 4.00 1.39 34.71

Pre-mR A cleavage

complex 2 protein Pcfl 1 PCF1 1 172.93 0.99 4 4.00 0.57 14.18

Pro tein DBF4 homo log A DBF4A 76.79 0.98 5 4.00 0.76 19.03

Putative heat shock protein

HSP 90-alpha A5 HS905 38.70 0.99 2 4.00 1.36 33.95

Rho GTPase-activating

protein 21 RHG21 217.18 0.97 4 4.00 0.37 9.18

Rho GTPase-activating

protein 35 RHG35 172.10 0.90 2 4.00 1.14 28.47

Ribosome-binding protein 1 RRBP1 152.36 1.00 5 4.00 0.92 22.97

S phase cyclin A-associated

protein in the endoplasmic

reticulum SCAPE 158.04 0.99 5 4.00 0.88 22.04

Schlafen family member 5 SLFN5 100.97 0.97 2 4.00 0.46 1 1.57

Sorbitol dehydrogenase DHSO 38.28 1.00 2 4.00 0.40 10.09

Synaptotagmin-1 SYT1 47.52 0.98 3 4.00 0.66 16.57

Zinc finger CCCH domain- containing protein 3 ^^_ 1"! 3 101.86 1.00 3 4.00 1.46 36.50

Zinc finger homeobox

protein 4 ZFHX4 393.46 0.76 3 4.00 0.86 21.41

Zinc finger MYM-type ZMYM

protein 4 4 172.66 0.94 3 4.00 1.39 34.86

Zinc finger protein 318 ZN318 250.94 1.00 7 4.00 1.21 30.19

Zinc finger protein 649 ZN649 57.63 0.91 2 4.00 0.61 15.33

Zinc finger protein Rlf RLF 217.80 1.00 6 4.00 0.61 15.36

Card iomy opathy-assoc iated

protein 5 CMYA5 448.92 1 .00 8 4.00 0.61 15.15

CpG-binding protein CXXC1 75.65 1.00 4 4.00 0.49 12.37

Keratin, type II cytoskeletal

74 K2C74 57.81 0.90 3 4.00 0.79 19.80

Protein CBFA2T2 MTG8R 67.07 1.00 4 4.00 0.50 12.62

UG0315GO3 Q8IXG5 23.16 0.88 2 4.00 0.46 1 1 .54

Coiled-coil domain- containing protein 41 CCD41 81.99 1.00 4 4.00 0.90 22.57

Dynein heavy chain 9,

axonemal DYH9 51 1.53 LOO 7 4.00 1.44 35.96

FCH domain only protein 2 FCH02 88.85 0.97 3 4.00 0.94 23.61

Intraflagellar transport

protein 74 homo log IFT74 69.18 0.99 2 4.00 0.71 17.81

Laminin subunit alpha-3 LAMA3 366.40 LOO 8 4.00 1.34 33.52

Proline-rich protein 12 PRR12 129.89 0.89 3 4.00 0.71 17.80

Ras-like protein family

member 1 1 A RSLBA 26.97 0.81 2 4.00 1.09 27.34

Ras-related protein Rab-35 RAB35 22.99 LOO 2 4.00 0.72 1 8.00 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa) Probability Guidelines) Intensity stdev stdev

Selenocysteine insertion

sequence-binding protein 2 SEBP2 95.38 0.98 4 4.00 1.09 27.29

Synaptonemal complex

protein 2 SYCP2 175.51 1.00 5 4.00 0.86 21.41

Synaptotagmin-like protein

2 104.85 0.87 2 4.00 1.05 26.35

Thyroid hormone receptor- associated protein 3 TR150 108.58 0.96 4 4.00 0.70 17.60 von Willebrand factor VWF 309.04 1.00 5 4.00 0.91 22.88

WD repeat-containing

protein 67 WDR67 124.09 1.00 5 4.00 1.24 31.13

Charged multivesicular

body protein 2a CHM2A 25.07 1.00 4 4.00 0.95 23.70

Liprin-beta-1 LIPB 1 1 13.93 0.96 3 4.00 1.19 29.82

Ras GTPase-activating

protein 1 RASA1 1 16.31 0.99 3 4.00 0.57 14.17

Zinc finger homeobox

protein 3 ZFHX3 404.15 1.00 5 4.00 0.59 14.84

A disintegrin and

metal loproteinase with

thrombospondin motifs 8 H0Y6F1 98.67 0.99 4 4.00 0.61 15.34

MYOl

Unconventional myosin-ih H 1 18.94 0.92 4 4.00 1.1 1 27.73

Apolipoprotein B

(Including Ag(X) antigen) C0JYY2 515.22 1.00 5 4,00 0.59 14.85

Proline-serine-threonine

phosphatase-interacting

protein 2 PPIP2 38,82 1.00 3 3.00 0.34 11.39

Xin actin-binding repeat- containing protein 2 XIRP2 382.05 0.94 3 3.00 1.73 57.52

A ctivity-regul ated

cytoskeleton-associated

protein ARC 45.27 0.97 2 3.00 0.54 17.86

Centrosome-associated

protein 350 CE350 350.70 0.98 4 3.00 0.48 16.14

Coiled-coil domain- containing protein

KIAA1407 K1407 110.48 0.99 2 3.00 1.59 52.91

Cytoskeleton-associated

protein 5 CKAP5 225.33 1.00 5 3.00 0.38 12.59

Disintegrin and

metalloproteinase domain- containing protein 12 ADA 12 99.46 1.00 4 3.00 0.55 18.43

E3 ubiquitin-protein ligase

HECTD1 HECD1 289.19 0.99 2 3.00 0.68 22.68

Endothe!in-1 EDN1 24.39 0.97 2 3.00 0.58 19.32

Fanconi-associated

nuclease 1 FAN1 1 14.13 0.98 3 3.00 0.74 24.65

Fibrinogen alpha chain FIBA 94.90 1.00 2 3.00 0.80 26.73

GTP-binding protein 1 GTPB1 72.39 1.00 4 3.00 0.82 27.23 Total

Protein Total Peptides

Weight GrouD (Paris % rel

Sequence Name Gene ID (kDa Probability Guidelines) Intensity stdev stdev

Mediator of RNA

polymerase II transcription

sub nit 14 MED 14 160.49 1.00 3 3.00 0.82 27.45

Mitotic checkpoint

serine/threonine-protein

kinase BUB1 beta BUB B 1 19.45 0.87 2 3.00 0.68 22.56

Nitric oxide-associated

protein 1 NOA1 78.39 0.82 2 3.00 0.87 28.88

Nucieosome-remodeling

factor subunit BPTF BPTF 338.04 0.94 4 3.00 0.56 18.54

Protein FAM208A F208A 188.90 0.97 3 3.00 0.50 16.51

Putative heat shock protein

HSP 90-beta 2 H90B2 44.30 0.96 4 3.00 0.66 22.09

Serine/threon i n e-protein

kinase Nekl NEK1 142.72 0.92 2 3.00 0.76 25.26

Tyrosine-protein kinase

BAZI B BAZIB 170.78 1 ,00 4 3.00 0.53 17.55

UBX domain-containing

protein 10 UBX 10 30.77 0.93 3 3.00 0.89 29.70

Unconventional myosin- MY09

IXa A 292.50 1.00 8 3.00 0.90 29.99

UPF0536 protein C12orf66 CL066 50.36 0.95 2 3.00 0.52 17.21

Vacuolar protein sorting- associated protein 13D VP13D 491.52 1.00 6 3.00 0.93 31 .07

Zinc finger CCHC domain- containing protein 24 ZCH24 26.92 0.89 2 3.00 1.29 43,02

Zinc finger protein 652 ZN652 69.68 0.99 6 3.00 1 ,27 42.20

Ankyrin Gl 19 Q13484 119.34 0.96 3 3.00 1.35 44.85

Elongation factor Ts,

mitochondrial EFTS 35.35 0.95 3 3.00 0.75 25.14

Erythrocyte band 7 integral

membrane protein STOM 1.69 0.93 2 3.00 0.45 14.92

Methyl cytosine

dioxygenase TET1 ^" TjST 1 235.14 0.99 4 3.00 0.90 30, 14

Testis -expressed sequence

9 protein TEX9 44.78 0.97 4 3.00 1.09 36.24

Trafficking protein particle

complex subunit 8 TPPC8 160.88 0.74 2 3.00 0.81 27.1 1

Transmembrane channellike protein 6 TMC6 89.97 0.90 2 3.00 0.16 5.41

Trichoplein keratin

filament-binding protein TCHP 61.02 0.99 3 3.00 0.81 27.04

Unconventional myosin- IXb MY09B 243.23 1.00 5 3.00 0.13 4,40

Zinc finger protein 485 ZN485 50.23 0.80 2 3.00 0.52 17.16

Zinc finger protein

basonuclin-1 BNC1 1 10.88 0.73 2 3.00 0.50 16.58

Coiled-coil domain- containing protein 81 CCD81 76.02 0.99 4 3.00 0.38 12.78

Sentrin-specific protease 2 SENP2 67.79 1.00 3 3.00 0.54 17.98 Total

Protein Total Peptides

Weight Group (Paris % rei

Sequence Name Gene ID fkDa) Probability Guidelines) intensity stdev stdev

Sorting nexin-33 SNX33 65.21 0.78 2 3.00 0.69 23.13

Ubiquitin carboxyl-terminal

hydrolase 20 UBP20 101.92 1.00 3 3.00 1 .12 37.50

7-dehydrocho lesterol

reductase DHCR7 54.44 0.91 2 3.00 0.88 29.46

ABI gene family, member 3

(NESH) binding protein,

tsoforrn CRA d Q5JPC9 110.59 1.00 4 3.00 1.08 35.95

Acid ceramidase subunit E7EMM

beta 4 41.75 0.99 2 3.00 0.27 9.06

Ankyrin repeat domain- containing protein 1 1 ANR11 297.71 0.99 5 3.00 0.62 20.58

Ankyrin repeat domain- containing protein 24 ANR24 124.09 1.00 4 3.00 1.02 34.05

ATP-binding cassette subfamily A member 13 ABCAD 575.77 0.99 4 3.00 0.90 30.14

AT-rich interactive domain- containing protein 5B ARI5B 132.27 1.00 5 3.00 0.96 32.12

Bone morphogenetic

protein receptor type- 1 A B R1A 60.14 0.73 2 3.00 0.58 19.23

Caicyphosin CAYP1 20.94 1.00 2 3.00 0.66 22.00

Ceil division cycle 5 -like

protein CDC5L 92.18 0.80 2 3.00 0.70 23.45

Centrosomal protein of 128

kDa CE128 127.92 0.96 3 3.00 0.88 29.30

B72 L

CHD9 protein 1 325.78 1.00 5 3.00 0.82 27.29

Chromosome 10 open

reading frame 10S, isoform Q5VX

CRA a 3 21.74 0.95 2 3.00 0.66 21.85

Cleavage and

polyadenylation specificity

factor subunit 7 CPSF7 52.00 0.99 3 3.00 1.51 50.17

Cluster in cms 52.44 LOO 4 3.00 0.59 19.55

Collagen alpha-2(IX) chain C09A2 65.07 1.00 4 3.00 1.24 41.49

Cone cGMP-specific 3 ',5'- cyclic phosphodiesterase

subunit alpha' PDE6C 99.07 0.76 2 3.00 1.1 1 37.13

Contactin-1 CNTN1 1 13.23 0.97 2 3.00 0.74 24.63

Cubilin CUBN 398.46 1.00 3 3.00 0.86 28.60

Cullin-associated NEDD8- dissociated protein 1 CAND1 136.27 0.98 4 3.00 0.75 25.04

DEN domain-containing

protein 5A DEN5A 146.98 1.00 2 3.00 0.89 29.50

DmX-like protein 2 DMXL2 339.41 0.94 3 3.00 0.60 19.85

DNA mismatch repair

protein Msh6 MSH6 152.67 0.97 4 3.00 0.69 22.85

DNA polymerase subunit

gamma- 1 DPOG1 139.46 0.85 2 3.00 0.26 8.75 Total

Protein Total Peptides

Weight Group fParis % rel

Sequence Name Gene ID (kDa) Probability Guidelines) Intensity stdev stdev

DNA polymerase zeta

catalytic subunit DPOLZ 352,54 0.91 3 3.00 0.22 7.42

DNA topoisomerase 3- aipha TOP3A 112.28 1.00 3 3.00 1.1 1 36.92

Dynein heavy chain 12,

axonemal DYH12 356.69 0.99 6 3.00 0.42 13.85

Dystonin DYST 860.1 1 1.00 1 1 3.00 0.56 18.71

Dystrophin DMD 426.47 1.00 8 3.00 1.03 34.32

E3 ubiquitin-protein ligase

BRE1 A BREIA 1 13.57 1.00 4 3.00 0.65 21 .62

E3 ubiquitin-protein ligase

BRE1 B BRE1B 1 13.56 1.00 5 3.00 1.15 38.20

E3 ubiquitin-protein ligase

HERC2 HERC2 526.88 0.79 3 3.00 0.47 15.69

E3 ubiquitin-protein ligase

UBR4 UBR4 573.46 0.99 3 3.00 0.97 32.37

EF-hand calcium-binding

domain-containing protein

6 EFCB6 172.80 1.00 6 3.00 0.45 15.1 1

Estrogen receptor ESR1 66.16 0.96 3 3.00 0.66 21.86

Exocyst complex

component 1 EXOC1 101.90 0.99 3 3.00 1.68 56.03

Extended synaptotagmin-3 ESYT3 99.95 1.00 4 3.00 0.61 20.41

Glutathione S-transferase

Mu 5 GSTM5 25.64 0.99 4 3.00 0.75 24.88

GRIP and coiled-coil

domain-containing protein

2 GCC2 195.77 0.99 3 3.00 0.62 20.55

GTPase KRas RASK 21.62 0.96 2 3.00 0.91 30.48

Guanine nucleotide-binding

protein G(t) subunit a!pha-1 GNAT1 40.00 0.80 2 3.00 0.81 27.16

Heterogeneous nuclear

ribonucleoprotein AO ROAO 30.80 0.98 2 3.00 1.15 38.44

Immunoglobulin-like and

fibronectin type III domain- containing protein 1 IGFN1 137.66 0.96 2 3.00 1.33 44.20

Insulinoma-associated

protein 2 INSM2 59.44 0.91 3 3.00 1.32 44.07

Inter-alpha-trypsin inhibitor

heavy chain HI ITIHl 101.31 0.99 2 3.00 0.95 31 .66

Interleukin-10 receptor

subunit alpha I10R1 62.95 0.91 4 3.00 0.50 16.69

Junctophilin-3 JPH3 81.40 0.91 3 3.00 0.99 32.95

Keratin, type I cytoske!etal

16 K1 C16 51.22 0.89 3 3,00 1.03 34.34

Keratin, type II cytoskeletal

7 K2C7 51.34 0.91 4 3.00 0.76 25.46

Kinesin-like protein

KIF13A KI13A 202.16 0.90 3 3.00 0.49 16.23

Krueppel-like factor 1 1 KLFU 55.09 0.87 2 3.00 0.13 4.30 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID fkDa) Probability Guidelines) Intensity stdev stdev

Leucine-rich repeat- containing protein 8C LRC8C 92.37 0.86 2 3.00 0.42 13.90

Leucine-rich repeat- containing protein 8E LRC8E 90.17 0.98 2 3.00 0.42 13.90

Ligand-dependent nuclear

receptor-interacting factor 1 LRIF1 84,50 1.00 6 3.00 0.43 14.48

LIM and calponin

homology domains- containing protein 1 LIMCl 121.77 0.95 2 3.00 0.65 21.66

Low density lipoprotein

receptor adapter protein 1 ARH 33.85 1.00 2 3.00 0,67 22.27

Lysophosphatidic acid

phosphatase type 6 PPA6 48.80 0.99 2 3.00 0.72 23.98

Lysosome-associated

membrane glycoprotein 1 LAMP1 44.84 1.00 2 3.00 0.60 20.04

Matrix extracellular

phosphoglycoprotein MEPE 58.37 0.90 3 3.00 0.44 14.65

Matrix-remodel ing- associated protein 5 MXRA5 31 1.94 0.96 3 3.00 0.54 17.94

Microtubule-associated

protein 4 MAP4 120.93 0.95 4 3.00 1.24 41.43

Midnolin MIDN 49.16 0.96 3 3.00 0.67 22.17

Monocarboxylate

transporter 7 MOT7 57.34 1.00 2 3.00 0.70 23.34

N-acetyltransferase ESC02 ESC02 68.25 0.98 4 3.00 0.45 15.05

Neurochondrin NCDN 78.80 0.93 2 3.00 0.99 33.14

Q5T3V

NOELI 3 VI 6 52.75 0.99 3 3.00 . 0.50 16.73

Nuclear pore complex

protein Nup88 NUP88 83.47 0.71 2 3.00 0.43 14.43

Nucleolar and coiled-body

phosphoprotein 1 NOLC1 73.54 0.99 2 3.00 1.28 42.79

Nucleolar protein 8 NOL8 131.52 0.99 3 3.00 0.60 19.98

Oxidoreductase HTATIP2 HTAI2 27.01 0.91 2 3.00 0.61 20.49

Partitioning defective 3

homolog B PAR3L 132.39 0.83 2 3.00 0.57 18.94

Pentatricopeptide repeat- containing protein 2 PTCD2 43.92 0.84 2 3.00 0.77 25.67

Pescadillo homolog PESC 67.94 0.84 2 3.00 0.85 28.42

Phosphate carrier protein,

mitochondrial MPCP 40.05 1.00 2 3.00 0.49 16.21

Plasma membrane calcium- transporting ATPase 1 AT2B 1 138.65 0.93 2 3.00 0.90 30.02

Plexin-Cl PLXC1 175.61 0.97 3 3.00 0.80 26.63

Protease serine 4 isoform B Q7Z5F4 28.16 0.97 2 3.00 0.76 25.29

Protein FAM186A F186A 262.58 0.99 4 3.00 0.45 14.84

Protein SMG8 SMG8 109.60 0.99 2 3.00 0.88 29.28

Protein TBRG4 TBRG4 70.68 0.99 4 3.00 0.43 14.50

Protein unc-80 homolog UNC80 363.14 0.90 4 3.00 0.58 19.40 Total

Protein Total Peptides

Weieht Group (Paris % rel

Sequence Name Gene ID fkDa) Probability Guidelines) Intensity stdev stdev

Putative HERC2-like

protein 3 HRC23 128.84 0.95 5 3.00 0.96 32.08

Rab proteins

geranylgeranyltransferase

component A 2 RAE2 74.01 0.83 3 3.00 1 .10 36.81

Ras GTPase-activating

protein 3 I J IS A 95.62 0.94 2 3.00 0.29 9.78

Ras-related protein Rab- 10 RABI O 22.51 1.00 4 3.00 0.87 28.91

Ribosomal protein S6

kinase, 70kDa, polypeptide Q9BRS

2 0 53.39 0.98 3 3.00 0.74 24.70

Ribosome biogenesis

regulatory protein homolog RRS 1 41.15 0.88 2 3.00 . 0.42 13.99

RING finger protein 169 RN169 77.13 0.82 2 3.00 1.14 38.3 0

Rootletin CROCC 228.37 1.00 7 3.00 1.37 45.54

Serine/threonine-protein

kinase MARK2 MARK2 87.84 1.00 4 3.00 0.82 27.35

SP-A receptor submit SP- Q5QD0

R210 alphaS ί 180.42 0.97 3 3.00 0.55 18.43

Splicing factor, suppressor

of white-apricot homolog SFSWA 104.74 0.99 2 3.00 0.40 13.35

Synaptonemal complex

protein 1 SYCPi 1 14.10 0.98 3 3.00 0.83 27.71

TBC1 domain family

member 9B TBC9B 140.42 0.98 2 3.00 0.29 9.71

THO complex subunit 2 THOC2 182.64 0.92 3 3.00 0.90 30.12

Thymosin beta- 10 TYB10 5.00 0.97 2 3.00 0.99 32.88

Transcriptional regulator Q6IEH8 315.85 0.99 5 3.00 1.25 41 .65

Transforming acidic coiled- coil-containtng protein 2 TACC2 309.22 0.98 5 3.00 0.75 24.92

Transmembrane channellike protein 2 TMC2 102.53 0.91 3 3.00 0.71 23.70

Tuberin TSC2 200.46 0.90 3 3.00 0.89 29.55

Ubiquitin carboxy!-terminal

hydrolase 24 UBP24 294.16 1 .00 4 3.00 0.67 22.50

Unconventional myosin- XV MY0 5 395.03 0.99 5 3.00 0.57 18.92

UPF0471 protein CI orf63 CA063 33.58 0.85 2 3.00 0.48 15.93

Voltage-dependent anion- selective channel protein 1 VDAC1 30.74 1 .00 4 3.00 0.93 31.16

Zinc finger protein 197 ZN197 118.75 1 ,00 5 3.00 0.80 26.83

Zinc finger protein 518A Z518A 166.66 0.99 4 3.00 0.50 16.71

Zinc finger protein 528 ZN528 72.07 1.00 2 3.00 0.81 26.88

Zinc finger protein 578 ZN578 42.55 0.98 2 3.00 0.53 17.73

Zinc finger protein 616 ZN616 90.20 1.00 4 3.00 0.46 15.29

Brefeldin A-inhtbited

guanine nucleotide- exchange protein 2 BIG2 201.89 0.98 4 3.00 1 22 40.70

Tumor suppressor p53- binding protein 1 TP53B 213.42 0.98 4 3.00 0.75 25.13 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene iD (kDa) Probability Guidelines Intensity stdev stdev

WASP homo log-associated

protein with actin,

membranes and WHAM

microtubules M 90.85 0.96 3 3.00 0.33 1 1.16

BAH and coiled-coil

domain-containing protein F8WB

1 W8 276.93 0.99 5 3.00 0.54 17.86

Complement Cl q tumor

necrosis factor-related

protein 4 C1 QT4 35.22 0.80 2 3.00 0.86 28,54

Dynein heavy chain 5,

axonemal DYH5 528.67 1.00 7 3.00 0.93 31.16

Ephrin type-B receptor 2 EPHB2 117.40 0.95 3 3.00 0.58 19,40

GON-4-like protein GON4L 248.45 0.99 4 3.00 0.81 27.16

Keratin, type I cuticular

Ha4 KRT34 49.37 0.77 2 3.00 0.47 15.75

Leuctne-rich repeat- containing protein 30 LRC30 33.99 0.99 2 3.00 0.51 16.87

Progesterone- induced- fa locking factor 1 PIBF1 89.73 0.99 4 3.00 0.58 19.19

Suppression of

tumorigenicity 18 protein ST18 1 15.06 1.00 3 3.00 1.26 42.12

Transient receptor potential

cation channel subfamily M

member 1 TRPM1 182.04 0.94 3 3.00 0.51 16,97

Unconventional myosin- MY07

VOa A 254.23 1.00 5 3.00 0.88 29.46

Voltage-dependent L-type

calcium channel subunit

alpha- I F CAC1F 220.52 0.89 3 3.00 1.20 40.00

Zinc finger protein 329 ZN329 61.67 1.00 3 3.00 0.42 14.10

Zinc finger protein 763 Z 763 46.05 0.86 2 3.00 0.93 30.97

60S ribosomal protein L24 RU24 17.75 0.95 3 3.00 0.84 28.12

Ankyrin repeat domain- containing protein 3 OB AN30B 157.93 0.99 4 3.00 0.58 19.50

Arginine vasopressin

receptor 1 Q3S2J4 46.81 0.99 3 3.00 1 .09 36.31

Bromodomain and WD

repeat-containing protein 1 BRWD1 262.75 1.00 9 3.00 1 .30 43.41

Cerebral protein- 12 Q96JS2 91.09 0.99 4 3.00 1.06 35.25

Collagen alpha-2(VI) chain C06A2 108.49 1.00 5 3.00 0.70 23.22

Collagen alpha-6(VI) chain C06A6 247.00 1.00 5 3.00 0.87 29.1 1

CUB and sushi domain- containing protein 3 CSMD3 405.72 0.95 3 3.00 0,63 20.94

E3 ubiquitin-protein ligase

SHPRH SHPRH 192.94 0.85 3 3.00 1.01 33.80

Endothelin-1 receptor EDNRA 48.67 1.00 2 3.00 0,57 19.01

Fibroblast growth factor 17 FGF17 24.86 0.81 2 3.00 0.61 20.49 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa Probability Guidelines Intensity stdev stdev

Histone-lysine N- methyltransferase

SUV420H1 SV421 99.1 1 LOO 5 3.00 0.96 31.92

Protein rnab-21-like 2 MB212 40.88 LOO 3 3.00 0.69 22.97

Protein S100-A6 S10A6 10.16 LOO 3 3.00 1.25 41.71

Regulating synaptic

membrane exocytosis

protein 3 RIM S3 32.76 0.96 2 3.00 0.25 8.38

Ribosomal protein S6

kinase a!pha-2 KS6A2 83.17 0.95 4 3.00 0.75 25.05

Taxi -binding protein 1 TAXB l 90.80 0.89 2 3.00 0.68 22.76

Thy-i membrane

glycoprotein THY1 17.91 LOO 2 3.00 0.52 17.25

Transformation/transcripiio

n domain-associated protein TRRAP 437.30 0.99 2 3.00 0.46 15.25

Zinc finger protein 536 ZN536 141.31 0.98 4 3.00 0.52 17.24

Zinc phosphodiesterase

ELAC protein 1 RNZ1 39.98 0.86 2 3.00 0.87 29.02

Zinc finger MYM-type ZMYM

protein 3 3 152.26 0.94 2 3.00 0.51 16.91

Tetratricopeptide repeat

protein 1 TTC1 33.49 0.97 2 2.00 0.76 37.78

A disintegrin and

metalloproteinase with

thrombospondin motifs 12 ATS 12 177.54 LOO 4 2.00 0.59 29.48

Alpha-N-acetyineuraminide

alph a-2, 8-s i aly ltrans ferase SIA8A 40.47 1.00 5 2.00 0.65 32.36

Ankyrin repeat domain- containing protein 32 ANR32 1.20.95 0.84 2 2.00 0.55 27.65

Chromodomain Y-Hke

protein CDYL1 66.42 0.99 2 2.00 0.99 49.53

Glutamyl-tRNA(Gln)

amidotransferase subunit A,

mitochondrial GATA 57.41 0.81 2 2.00 0.37 18.58

Hepatocyte growth factor

receptor MET 155.42 1.00 4 2.00 0.47 23.48

Histone H3.3C H3C 15.19 0.97 3 2.00 0.58 29.20

Hydrocephalus-inducing

protein homoiog HYDIN 575.51 0.99 3 2.00 0.36 18.22

Insulin receptor INSR 156.21 1 .00 5 2.00 0.85 42.64

Kinesin-like protein KIF15 KIF15 160.04 0.81 2 2.00 0.86 43.07

La-related protein 1 LARP1 123.42 0.92 3 2.00 0.40 20.15

Leucine-rich PPR motif- containing protein,

mitochondrial LPPRC 157.79 0.99 4 2.00 0.64 31.73

Mesothelin MSLN 68.92 0.94 2 2.00 0.49 24.57

Q8NEW

p53-activated protein-2 2 19.22 0.81 2 2.00 0.46 23.21

PH and SEC7 domain- containing protein 2 PSD2 84.59 0.83 2 2.00 0.69 34.26 j Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID fkDa) Probability Guidelines) Intensity stdev stdev

Polycystin-2 P D2 109,60 1 ,00 4 2.00 0.65 32.31

Protein prune homolog 2 PRUN2 340.41 0.99 2 2.00 0.53 26.26

Protocadherin-16 PCD 16 345.95 0.77 2 2.00 0.21 10.31

SEC 14 domain and spectrin

repeat-containing protein 1 SESD1 79.28 0.98 4 2.00 0.19 9.64

Spectrin beta chain,

erythrocyte SPTB1 246.30 0.96 3 2.00 0.73 36.41

Sterile alpha motif domain- containing protein 9-1 ike SAM9L 184,40 0.89 4 2.00 0.66 32.84

Testis-specific HI histone H1FNT 28.08 0.93 4 2.00 0.33 16.30

Zinc finger with UFM1- specific peptidase domain

protein ZUFSP 65.90 0.79 2 2.00 0.68 34.15

A disintegrin and

metalloproteinase with B7ZVX

thrombospondin motifs 9 9 213.26 1.00 8 2.00 0.77 38.37

Actin-binding protein

antllin ANLN 124.10 1 .00 2 2.00 0.39 19.35

AF4/FMR2 family member

3 C9JXV5 109.90 0.87 4 2.00 0.56 27.89

Ankyrin repeat domain- containing protein 7 ANKR7 28.99 0.81 2 2.00 0.51 25.50

Antigen KI-67 KI67 358.46 1.00 9 2.00 0.49 24.25

AT-rich interactive domain- containing protein 1A ARI1A 241.87 0.99 7 2.00 0.79 39.29

BCL-6 corepressor BCOR 192.05 0.95 5 2,00 0.73 36.27

BCL6 corepressor/retinoic

acid receptor alpha fusion E3UVQ

protein 2 21 1.05 0.84 5 2.00 0.73 36.27

Brain-specific angiogenesis

inhibitor -associated

protein 2-like protein 1 BI2L1 56.83 1.00 2 2.00 0.43 21.28

Brefeidin A-inhibited

guanine nuc!eotide- exchange protein 1 BIG1 208.62 0,98 3 2.00 0.49 24.61

C4b-binding protein beta

chain C4BPB 28.32 0.83 2 2.00 0.51 25.44

Chondroitin sulfate N- acetylgalactosaminyltransfe

rase 1 CGAT1 61.24 0.99 2 2.00 0.48 23.94

Chromodomain-helicase-

DNA-binding protein 7 CHD7 335.70 0.99 5 2.00 0.65 32.72

Citron Rho-interacting

kinase CTRO 231.27 1.00 6 2.00 0,75 37.35

Coiied-coil domain- containing protein 40 CCD40 130.02 1.00 3 2.00 0.50 24,76

CulIin-3 CUL3 88.86 0.81 2 2.00 0.83 41.66

Cycl in-dependent kinase 13 CD 13 164.80 LOO 7 2,00 0.66 32.80

Deubiquitinating protein

VCIP135 VCIP1 134,22 0.80 2 2.00 0.42 20.81 Total

Protein Total Peptides

Weight Group (Paris % rel

Seauence Name Gene ID (kDa) Probability Guidelines) Intensity stdev stdev

FACT complex subunit

SPT16 SP16H 1 19.82 0.98 4 2.00 0.61 30.50

FERM and PDZ domain- containing protein 4 FRPD4 144.27 0.87 4 2.00 0.78 39.04

Fibroblast growth factor 1 1 FGF11 24.97 0.89 3 2.00 0.61 30.61

Gamma-interferon- inducible protein 16 IF16 88.18 0.83 2 2.00 0.33 16.55

GTP-binding protein GEM GEM 33.91 0.97 3 2.00 0.76 38.19

Interphotoreceptor matrix

proteoglycan 2 IMPG2 138.52 0.92 3 2.00 0.31 15.59

JunctophiIin-2 JPH2 74.16 0.70 2 2.00 1.07 53.37

Kal!ikrein-5 KLK5 31.98 0.74 2 2.00 0.61 30.60

Keratin, type ΪΙ cuticular

Hb3 KRT83 54.14 1.00 3 2.00 0.57 28.45

Keratin, type II cytoskeletal

2 epidermai K22E 65.38 0.70 3 2.00 0.62 31.19

Leucine-rich repeat

transmembrane neuronal

protein 2 LRRT2 59.02 0.75 2 2.00 0.58 29.1 1

Leucine-rich repeat- containing protein 45 LRC45 75.89 1.00 3 2.00 0.55 27.52

Macoi!in MACOI 76.1 1 1.00 3 2.00 1.13 56.48

Malate dehydrogenase,

cytoplasmic MDHC 36.39 0.97 5 2.00 0.42 21.10

Meiosis-specific nuclear

structural protein 1 MNS 1 60.52 0.97 3 2.00 0.83 41.63

Membrane protein,

palmitoylated 5 (MAGUK Q4VBP

p55 subfamily member 5) 4 77.26 LOO 3 2.00 0.41 20.37

Myosin- 15 MYH35 224.46 LOO 7 2.00 0.66 33.00

NF-kappa-B inhibitor

epsilon IKBE 52.81 0.83 2 2.00 0.65 32.59

Q5H9C

Novel protein 3 33.04 0.98 2 2.00 0.36 17.80

Nuclear body protein

SP140-like protein LY10L 66.94 0.98 3 2.00 0.42 21.13

Nuclear protein localization

protein 4 hornolog NPL4 68.06 1.00 3 2.00 0.39 19.73

Peptidyl-prolyl cis-trans

isomerase G PPIG 88.55 0.98 4 2.00 0.60 30.10

Peroxisome proliferator- activated receptor gamma

coactivator-reiated protein

1 PPRC1 177.42 0.99 6 2.00 0.80 39.94

PHD finger protein 20 PHF20 1 15.30 0.98 2 2.00 1.04 52.12

A0AV3

Plakophilin 2 7 92.68 LOO 4 2.00 0.26 12.99

Protein AHNAK2 AHNK2 616.22 0.96 4 2.00 0.92 46.10

Protein BTG3 BTG3 29.08 0.73 2 2.00 0.61 30.69 Total

Protein Total Peptides

Weight Group (Paris % rel

Seauence Name Gene ID fkDa Probability Guidelines) Intensity stdev stdev

Protein phosphatase

Slingshot homolog 1 SSH1 1 15.42 0.86 3 2.00 0.76 37.80

Rabenosyn-5 RBNS5 88.80 0.87 3 2.00 0.66 32.95

Receptor tyrosine-protein

kinase erbB-4 ERBB4 146.69 0.90 4 2.00 0.72 35.78

Ribosome biogenesis

protein NSA2 horaolog NSA2 30.03 0.81 2 2.00 0.39 19.38

RNA -binding protein 26 RBM26 1 13.51 0.97 4 2.00 0.45 22.70

Scaffold attachment factor

B l SAFB 1 102.56 0.91 3 2.00 0.57 28.72

SH3 and multiple ankyrin

repeat domains protein 2 SHAN2 158.71 0.96 3 2.00 0.45 22.63

Signal-induced

proliferation-associated 1- like protein 2 SI1 L2 190.30 0.89 2 2.00 1.29 64.66

Tetratricopeptide repeat

protein 21 A TT21A 150.83 0.98 2 2.00 0.35 17.37

Tigger transposable

element-derived protein 2 TIGD2 59.57 1.00 3 2.00 0.53 26.67

Torsin family protein

C9orfl 67 CI167 46.87 0.98 4 2.00 0.58 29.1 1

Transcription factor MafF MAFF 17.73 0.99 2 2.00 0.34 17.06

U4 U6 small nuclear

ribonucleoprotein Prp3 PRPF3 77.46 0.98 3 2.00 0.64 31.78

Usherin USH2A 575.21 0.94 3 2.00 0.33 16.50

Werner syndrome ATP- dependent helicase WRN 162.34 1 .00 7 2.00 0.68 34.06

Zinc finger protein 407 ZN407 247.19 0.93 3 2.00 0.73 36.71

Zinc finger protein 821 ZN821 46.68 0.86 2 2.00 0.44 22.16

Alcohol dehydrogenase

class 4 mu/sigma chain ADH7 41.44 0.98 3 2.00 0.71 35.37

AP-3 complex subunit E7EMM

delta- 1 2 11 1.16 1.00 2 2.00 0.40 20.19

Bromodomain adjacent to

zinc finger domain, 1A,

isoform CRA c D3DS96 178.44 1.00 3 2.00 0.79 39.48

Cartilage intermediate layer

protein 1 G-I J3? 1 132.46 1 .00 7 2.00 0.38 1 8.82

Cycl in-dependent kinase 9 CDK9 42.73 1 .00 3 2.00 0.82 40.94

Dedicator of cytokinesis

protein 6 DOC 6 229.40 0.98 3 2.00 0.32 15.82

DOCK4 protein Q149N2 127.43 0.99 3 2.00 0.81 40.75

Dynein heavy chain 1 1 ,

axonemal DYH 1 1 520.69 0.99 3 2.00 0.61 30.34

Hi stone Hloo H1 FOO 35.77 0.98 3 2.00 0.27 13.41

Integrin alpha- 1 ITA1 130.75 1 .00 4 2.00 0.47 23.58

Leucine-rich repeat and

calponm homology

domain-containing protein

1 LRCH1 80.81 0.94 3 2.00 0.55 27.45

family A member 5 3 186.44 0.99 4 2.00 0.19 9.64 Total

Protein Total Peptides

Weit;ht Group (Paris % rel

Sequence Name Gene ID fkDa) Pro ability Guidelines) : Intensity stdev stdev

AT-rich interactive domain- containing protein 4A ARI4A 142.65 0.85 2 2.00 0.34 17.17

A2RRM

BBX protein 7 71.42 0.95 4 2.00 0.21 10.73

B7ZM2

C9orf93 protein 2 153.65 0.90 2 2.00 0.39 19.51

Calcium-activated

potassium channel subunit

alpha- 1 KCMA1 137.45 0.96 2 2.00 0.47 23.64

E7ESM

Calpastatin 9 82.37 0.99 4 2.00 0.65 32.72

Carbonyl reductase A8MT

f ADPH] 1 Ml 24.46 0.84 2 2.00 0.25 12.46

Carnosine synthase 1 CRNS1 88.41 0.99 3 2.00 0.25 12.29

Centrosomal protein of 290

kDa CE290 290.19 0.96 2 2.00 0.96 47.76

Charged multivesicular

body protein 4b CHM4B 24.92 0.89 2 2.00 1.22 61.19

Chromosome-associated

kinesin KIF4A KIF4A 139.78 0.75 4 2.00 0.39 19.26

Chromosome-associated

kinesin KIF4B KIF4B 139.93 0.99 6 2.00 0.39 19.26

Coiied-coil domain- containing protein 121 CC121 33.02 0.97 2 2.00 0.79 39.64

Coiled-coil domain- containing protein 127 CC127 30.80 0.93 3 2.00 0.59 29,54

Coiled-coil domain- containing protein 168 CC168 277.76 1.00 5 2.00 0.64 31.98

Coiled-coil domain- containing protein 66 CCD66 109.33 1.00 6 2.00 0.99 49.65

Coi!ed-coil domain- containing protein 7 CCDC7 55.73 0.85 3 2.00 0.48 23.80

Coronin-7 COR07 100.52 0.99 4 2.00 0.64 31.91

Creatine kinase U-type,

mitochondrial CRU 46.99 0.97 2 2.00 0.16 8.01

Cytochrome P450, family

1 , subfamily B, polypeptide Q5TZV

1 6 60.78 0.99 2 2.00 0.44 21.82

DCNl -like protein 2 DCNL2 30.14 0.86 2 2.00 0.58 29.19

Dedicator of cytokinesis

protein 8 DOCKS 238.36 LOO 3 2.00 0.72 36.24

Diacylglycerol kinase iota DGKI 1 16.90 0.98 3 2.00 0.26 12.88

Dihydropyrimidine

dehydrogenase [NADP(+)] DPYD 11 1.31 0.90 3 2.00 0.75 37.38

Discs, large homolog 7

(Drosophiia), isoform A8MT

CRA c M6 94.10 0.97 3 2.00 0.50 25.07

DNA he!icase ΪΝΟ80 ΓΝΟ80 176.63 0.99 5 2.00 0.77 38.25

DNA repair protein

complementing XP-C cells XPC 105.87 0.77 2 2.00 0.28 14.1 1 Total

Protein Total Peptides

Weight Group ( ar is % rei

Sequence Name Gene ID (kDa) Probability Guidelines) Intensitv stdev stdev

DNA repair protein RAD50 RAD50 153.78 0.99 4 2.00 0.66 32.79

DNA-binding protein

SATB 1 SATB 1 85.89 0.96 3 2.00 0.90 45.19

DNA-directed RNA

polymerase I subunit RPAl R Al 194.67 1 ,00 4 2.00 0.89 44.67

DNA-directed RNA

polymerase I subunit

RPA49 RPA49 53.91 1.00 2 2.00 0.42 20.82

DNA-directed RNA

polymerase III subunit

RPC3 RPC3 60.56 0.81 2 2.00 0.66 32.85

Dual 3 ',5 '-cyclic- AMP and

-GMP phosphodiesterase

11A PDE1 1 104.67 0.94 3 2.00 1.16 58.11

Dual specificity tyros ine- phosphorylation-regulated

kinase 4 DYRK4 59.55 0.89 2 2.00 0.54 27.15

E3 ubiquitin-protein ligase

RNF8 RNF8 55.46 0.81 2 2.00 1.04 52.10

ELKS/Rab6- interacting/CAST family

member 1 RB6I2 127.99 0.98 4 2.00 0.45 22.34

Epidermal growth factor

receptor kinase substrate 8 EPS 8 91.81 1.00 4 2.00 0.63 30.74

Eukaryotic translation

initiation factor 3, subunit

A Q24JU4 166.35 1.00 6 2.00 1.06 52.98

Fatty acid synthase FAS 273.24 0.89 2 2.00 0.70 34.92

Fibroblast growth factor 13 FGF13 27.53 0.99 3 2.00 0.41 20.68

FK506-binding protein 15 F B15 133.53 0.93 2 2.00 0.83 41.63

G protein-coupled receptor

kinase 4 GRK4 66.52 0.71 2 2.00 0.24 1 1.97

Gl/S-specific cyclin-E2 CCNE2 46.71 0.99 4 2.00 0.57 28.68

Gamma-am inobutyric acid

receptor subunit rho-3 GBRR3 54.22 0.99 3 2.00 0.56 28.09

A6H8W

GIGYF2 protein 152.29 0.99 3 2.00 0.82 40.77

Glial fibrillary acidic

protein GFAP 49.83 0.87 3 2.00 0.59 29.72

Golgin subfamily A

member 3 GOGA3 167.23 0.98 4 2.00 0.43 21.34

Golgin subfamily B

member 1 GOGB 1 375.77 1.00 6 2.00 0.63 31.38

Heat shock protein 105 kDa HS105 96.79 0.72 2 2.00 0.69 34.46

Hepatocyte growth factor- regulated tyrosine kinase

substrate HGS 86.12 0.90 3 2.00 0.68 33.89 Total

Protein Total Peptides

Weight Group ( Par is % rel

Sequence Name Gene ID Probability Guidelines) Intensity stdev stdev

High mobility group

nucleosorae-binding

domain-containing protein HMGN

3 3 10.64 0.83 2 2.00 0.93 46.53

Histone H2A type 2-B H2A2B 13.97 0.91 2 2.00 0.58 29.24

Histone-lysine N- methyltransferase EHMT1 EHMTl 141.36 1.00 5 2.00 0.47 23.42

Histone-lysine N- rnethyitransferase SETDIA SET! A 185.90 0.86 2 2.00 0.49 24.28

Homeobox protein cut-like

1 CUX1 164.07 0.73 2 2.00 0.48 24.01

HormonaJly up-regulated

neu tumor-associated

kinase HUNK 79.62 0.82 2 2.00 0.43 21.39

Inactive dual specificity

phosphatase 27 DUS27 130.08 0.88 3 2.00 1.15 57.42

Inositol 1 ,4,5-trisphosphate

receptor type 1 ITPR1 313.73 0.83 2 2.00 0.70 35.15

Inositol hexakisphosphate

and diphosphoinositol- pentakisphosphate kinase 2 VIP2 140.30 0.90 2 2,00 0.42 21.07 insulin receptor substrate 4 IRS4 133.67 0.94 4 2.00 0.55 27.38 interleukin-l receptor

accessory protein IL1AP 65.36 0.80 2 2.00 0.59 29.35

Kalirin KALRN 339.94 1.00 8 2.00 0.83 41.56

Keratin, type H cuticular

Hb4 KRT84 64.78 0.93 3 2.00 0.66 33,20

Laminin subunit alpha- 1 LAMA1 336.85 0.99 6 2.00 0.38 18.87

Laminin subunit gamma- 1 LAMC1 177.47 0.72 2 2.00 0.21 10.39

Long-chain fatty acid

transport protein 3 S27A3 78.58 0.99 2 2.00 0.28 13.75

Mediator of R A

polymerase II transcription

subunit 13 MED 13 239.13 1.00 3 2.00 0.40 19.87

Metal transporter CNNM2 CNNM2 96.54 0.99 3 2.00 0.48 23.92

MICAL-like protein 1 MILK1 93.37 0.86 2 2.00 0.35 17.47

Mitochondrial import

receptor subunit TOM 4 TOM34 34.52 0.71 2 2.00 0.57 28.49

Mitochondrial

transmembrane GTPase

FZO-2 Q4AEJ4 86.92 0.91 3 2.00 0.38 19.03

Myosin light chain kinase

2, skeletal/cardiac muscle MYLK2 64.63 0.82 2 2.00 1.14 57.00

Myotonin-protein kinase DMPK 69.32 0.97 2 2.00 0.55 27.54

Neurabin-2 NEB2 89.12 0.99 4 2.00 0.74 37.05

Neurexin-2-alpha NRX2A 1 84.85 0.93 4 2.00 0.51 25.74

Neuroblast differentiation- associated protein AHNAK AHNK 628.68 0.96 5 2.00 0.31 15.38

Neuronal migration protein

doublecortin DCX 49.27 0.95 2 2.00 0.45 22.27 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID ikDa) Probability Guidelines) Intensity stdev stdev

N-iysine methyitransferase

SETD6 SETD6 53.14 0.96 2 2.00 0.76 38.21

One cut domain family

member 3 ONEC3 49.99 1.00 3 2.00 0.38 19.10

Pancreatic !ipase-related

protein 2 LIPR2 51.90 0.75 2 2.00 1.04 51.79

Patched domain-containing

protein 1 PTHD1 101.26 0.86 2 2.00 1.16 57.83

Peripherin PERI 53.60 0.97 2 2.00 0.21 10.68

Phosphatase and actin

regulator 3 PHAR3 62.50 0.94 2 2.00 0.34 17.12

Plectin PLEC 531.45 1.00 6 2.00 0.63 31.63

Polyamine-modulated

factor -binding protein I PMFBP 1 18.94 1.00 4 2.00 0.52 25.80

Polycystic kidney and

hepatic disease 1 Q86Z26 446.39 0.94 3 2.00 0.92 45.77

Probable ATP-dependent

RNA he!icase DDX49 DDX49 54.17 1 .00 3 2.00 0.75 37.37

Probable G-protein coupled

receptor 158 GP158 135.39 0.88 3 2.00 0.29 14.34

Protein bicaudal C homo log

1 BICC1 104.76 0.99 3 2.00 0.43 21.57

Protein downstream

neighbor of Son DONS 62.69 0.97 3 2.00 0.77 38.44

Protein FAM181 A F181A 38.68 1.00 3 2.00 0.55 27.65

Protein FAM55D FA55D 62.20 0.73 2 2.00 0.81 40.57

Protein GPRI 08 GP108 60.58 0.82 2 2.00 0.75 37.57

Protein IAA1 199 K1 199 152.88 0.85 2 2.00 0.62 31.02

Protein LSM14 homo log B LS14B 42.03 0.93 2 2.00 0.80 40.13

Protein numb homolog NUMB 70.74 1.00 3 2.00 0.46 22.93

Protein NYNRIN NYNRI 208.22 1.00 3 2.00 0.63 31.61

Protein phosphatase 1

regulatory subunit 26 PPR26 127.26 1.00 6 2.00 0.50 24.79

Protein PRR14L PR14L 237.13 0.96 3 2.00 0.65 32.54

Protein S100-A14 S10AE 1 1.64 1.00 3 2.00 0.77 38.72

Protein S100-P SI OOP 10.38 1.00 2 2.00 1.1 1 55.61

Protein SCAF8 SCAF8 140.42 0.92 2 2.00 0.43 21.73

Protein Shroom3 SHRM3 216.71 1.00 4 2.00 1.19 59.34

Protein SON SON 263.65 1.00 7 2.00 0.73 36.60

Protein strawberry notch

homolog 1 SBNOl 154.20 0.86 2 2.00 0.63 31.46

Protein unc-13 homolog A UN13A 192.87 0.95 4 2.00 1.33 66.72

Protein unc-45 homolog B UN45B 103.65 0.96 4 2.00 0.43 21.44

Protein WWC2 WWC2 133.79 1.00 5 2.00 0.63 31.28

Protein-methionine

sulfoxide oxidase MICALl MICA1 1 17.78 0.81 2 2.00 0.46 23.1.7

Putative ankyrin repeat

domain-containing protein

ENSP00000383069 YN01 1 21 ,32 0.99 2 2.00 0.41 20.52

Putative helicase MOV-10 MOV10 113.58 0.99 2 2.00 0.74 37.04 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa> Probability Guidelines} Intensity stdev stdev

Putative protein

FAM90A12P F90AC 49.74 0.83 2 2.00 0.76 37.90

Putative zinc finger protein

355P Z 55P 49.64 0.82 3 2.00 0.33 16.60

RasGAP-activating-Hke

protein 1 RASL1 89.94 0.99 2 2.00 0.62 30.97

Receptor-type tyrosine- protein phosphatase T PTPRT 162.01 0.98 3 2.00 0.94 47.20

Regulator of G-protein

signaling 21 RGS21 17.64 0.83 ₂ 2.00 0.26 13.23

Retinol dehydrogenase 1 1 RDH3 1 35.35 0.87 2 2.00 0.86 42.88

Rho GTPase- activating

protein 20 RHG20 132.51 0.95 4 2.00 0.31 15.63

Rho GTPase-activating

protein 33 RHG33 137.1 1 0.79 2 2.00 0.72 35.97

Rho GTPase-activating

protein 5 RHG05 172.33 0.93 3 2.00 0.93 46.64

Rho guanine nucleotide

exchange factor 40 ARH40 164.54 0.99 4 2.00 1.00 50.05

RING finger protein 145 RN145 75.55 0.84 2 2.00 0.51 25.47

RING1 and YY1 -binding

protein RYBP 24.79 0.97 3 2.00 1.14 56.90

RNA polymerase II

elongation factor ELL ELL 68.20 0.99 3 2.00 0.61 30.60

RNA polymerase II subunit

A C-terminal domain

phosphatase CTDP1 104.32 1.00 4 2.00 0.32 15.87

RNA polymerase II

transcription factor 8 III

subunit A2 ELOA2 83.85 0.97 2 2.00 0.70 34.98

Round spermatid basic

protein 1 -like protein RSBNL 94.79 0.89 3 2.00 0.78 38.77

RUN and FYVE domain- containing protein 1 RUFY1 79.75 0.97 2 2.00 0.31 15.38

Serine/threonine-protein

kinase mTOR MTOR 288.69 1.00 2 2,00 0.37 18.51

Serine/threonine-protein

kinase NIM1 NIM1 49.56 0.98 2 2.00 0.82 41.1 1

Serine/threonine-protein

kinase PLK1 PLK 1 68.19 0.98 3 2.00 0.48 24.08

Serum response factor- binding protein 1 SRFB1 48.59 0.98 2 2.00 0.35 17.54

Short transient receptor

potential channel 1 TRPC1 91.13 0.94 2 2.00 0.50 24.95

Sodium-dependent

dopamine transporter SC6A3 68.43 1 ,00 2 2.00 0.79 39.43

Spectrin beta chain, brain 4 SPTN5 416.56 1.00 7 2.00 0.63 31.63

Sperm-associated antigen 5 SPAG5 134.32 0.99 2 2.00 0.94 47.00

StAR-related lipid transfer

protein 8 STARS 1 12.51 0.96 2 2.00 0.33 16.39 Total

Protein Total Peptides

Weight Group (Paris % rei

Sequence Name Gene ID fkDa) Probability Guidelines) Intensity stdev stdev

Superkiller viralicidic

activity 2-like 2 SK2L2 1 17.71 0.76 2 2.00 0.68 34.17

Superoxide dismutase [Cu- Znl SODC 15.91 0.99 2 2.00 0.38 19.03

Supervillin SVIL 247.57 1.00 4 2.00 1.15 57.32

Suppressor of cytokine

signaling 4 SOCS4 50.57 0.97 3 2.00 0.64 31.90

Sushi domain-containing

protein 5 SUSD5 67.96 0.85 2 2.00 0.15 7.74

Telomere length regulation

protein TEL2 homolog TEL02 91.67 0.98 2 2.00 0.56 27.93

Testis-specific

chromodomain protein Y 1 CDYI 60.42 0.81 2 2.00 0.32 16.24

Threonine—tR A iigase,

cytoplasmic SYTC 83.36 0.88 2 2.00 0.20 9.90

Thrombospondin type-1

domain-containing protein

7B THS7B 179.27 0.88 3 2.00 0.58 28.80

Toll-like receptor 7 TLR7 120.83 0.85 3 2.00 0.63 31.55

Transcription elongation

regulator 1 TCRG1 123.81 0.86 3 2.00 0.30 14.95

Transcription intermediary

factor 1 -beta TIF IB 88.48 1.00 3 2.00 0.27 13.61

Transitional endoplasmic

reticulum ATPase TERA 89.25 0.94 ₂ 2.00 0.63 31.49

Transmembrane and TPR

repeat-containing protein 3 TMTC3 103.92 1.00 4 2.00 0.60 29.79

Triadin TRDN 81.53 0.94 2 2.00 0.79 39.25

Triosephosphate isomerase 30.75 1.00 3 2.00 0.92 46.20

Tropomyosin alpha-3 chain TPM3 32.78 1.00 4 2.00 0.87 43.51

Tudor domain-containing

protein 1 TDRD1 131.92 0.99 2 2.00 1.33 66.34

T infiiin-2 TWF2 39.51 0.99 4 2.00 0.68 34.08

Tyrosine-protein

phosphatase non-receptor

type 21 PTN21 133.18 0.99 5 2.00 0.95 47.42

Ubiquitin carboxyl-terminal

hydrolase 16 UBP1 93.49 0.97 2 2.00 0.64 31.91

Unc-45 homolog A (C.

elegans), isoform CRA a A8 6F7 101.59 1.00 3 2.00 0.68 34.05

Utrophin UTRO 394.20 0.99 3 2.00 0.89 44.70

Wings apart-like protein

homolog WAPL 132.84 0.94 2 2.00 0.52 26.09

Zinc finger and BTB

domain-containing protein

17 ZBT17 87.85 0.90 2 2.00 0.24 12.19

Zinc finger and SCAN

domain-containing protein

5A ZSA5A 55.81 0.84 2 2.00 0.60 30.02 Total

Protein Total Peptides

Weight Group (Paris % re!

Sequence Name Gene ID tkDa) Probability Guidelines) Intensity stdev stdev

Zinc finger BED domain- containing protein 4 ZBED4 130.22 0.92 2 2.00 0.54 26.92

Zinc finger C3H1 domain- containing protein ZC3H1 226.20 0.89 3 2.00 0.56 28.13

Zinc finger CCCH domain- containing protein 1 1 A 2C1 1 A 89.06 0.89 2 2.00 0.86 42.77

Zinc finger CCHC domain- containing protein 4 ZCHC4 58.95 0.89 3 2.00 0.44 22.23

Zinc finger imprinted 2 ZIM2 61.11 0.99 5 2.00 0.47 23.46

Zinc finger MYM-type ZMYM

protein 2 2 154.79 0.97 4 2.00 1.12 55.82

Zinc finger protein 217 ZN217 1 15.18 0.99 3 2.00 0.38 19.20

Zinc finger protein 230 ZN230 54.49 0.81 2 2.00 0.57 28.46

Zinc finger protein 267 ZN267 87.30 0.87 2 2.00 0.89 44.72

Zinc finger protein 62

homolog ZFP62 102.43 0.98 2 2.00 0.48 24.05

Zinc finger protein 804A Z804A 136.78 0.92 3 2.00 0.59 29.48

Zinc finger protein ZFAT ZFAT 138.93 1.00 5 2.00 0.88 44.19

Zinc finger SWIM domain- containing protein

KIAA0913 K0913 197.16 1.00 5 2.00 0.41 20.64

A-kinase anchor protein 12 AKA12 191.35 0.98 3 2.00 0.97 48.67

Amiloride-sensitive sodium

channel subunit alpha B4E2Q5 78.17 1.00 2 2.00 0.45 22.55

C8orf58 protein Q7Z758 35.78 1.00 3 2.00 0.42 21.19

Chromosome alignment- maintaining phosphoprotein

1 CHAP! 89.02 0.84 2 2.00 0.16 8.15

Ephrin type-A receptor 8 EPH 8 110.91 1.00 3 2.00 0.97 48.55

Epidermal growth factorlike protein 6 EGFL6 61.26 0.89 2 2.00 0.84 41.81

GBF1 protein Q149P0 205.87 0.99 5 2.00 0.28 13.79

HDCMC04P Q9NS29 51.86 0.88 4 2.00 0.29 14.55

■ Immunoglobulin

superfamily member 8 IGSF8 64.98 0.96 2 2.00 0.82 40.82

LIM domain only protein 7 LM07 192.56 1.00 6 2.00 0.91 45.56

Low-density lipoprotein

receptor-related protein 3 LRP3 82.81 0,94 2 2.00 0.56 27.92

Lysine-specific histone KDMI

demethylase 1A A 92.83 1.00 2 2.00 0.70 34.80

Mediator ofRNA

polymerase II transcription

subunit 12- like protein MD12L 239.95 0.96 3 2.00 0.37 18.69

Mitogen-activated protein

kinase- inding protein 1 MABP1 163.70 0.84 2 2.00 0.35 17,27

Nik-related protein kinase NR 178.35 1.00 3 2.00 0.61 30.40

PDZ domain-containing

RING finger protein 4 PZRN4 1 17.01 0.88 3 2.00 0.62 30.99

Q71RB

PP7302 0 23.02 0.99 2 2.00 0.35 17.29 Total

Protein Total Peptides

Weight Group (Paris % re!

Sequence Name Gene ID (kDa) Probability Guidelines ^' ) Intensity stdev stdev

Pre-mRNA-processing- splicing factor 8 PRP8 273.41 0.99 5 2.00 0.53 26.34

Rho GTPase-activating

protein 10 RHG10 89.30 0.87 2 2.00 0.63 31.56

Serine/arginine repetitive

matrix protein 1 SRRMl 102.26 1.00 8 2.00 0.33 16.72

Transmembrane protein

C19orf77 CS077 14.94 0.99 2 2.00 0.51 25.73

Treacle protein TCOF 152.00 1.00 4 2.00 0.42 20.96

V-type proton ATPase

subunit C 1 VATC1 43.90 0.95 3 2.00 0.54 27.10

WD repeat, SAM and U- box domain-containing

protein 1 WSDU1 52.76 0.77 2 2.00 0.56 27.92

Ankyrin-2 ANK2 429.97 0.95 4 2.00 1.25 62.44

ATPase family AAA

domain-containing protein

2 ATAD2 158.44 1.00 4 2.00 0.51 25.55

Histone-lysine N- methyltransferase ASH1L ASH1L 332.56 0.99 3 2.00 0.54 27.06

Leucine-rich repeat- containing protein

PRAME-like PRAML 58.1 1 0.99 2 2.00 0.74 37.24

Ribosome biogenesis

protein BMS 1 homolog BMS 1 145.70 0.93 2 2.00 0.89 44.45

Testis -specific gene 30

protein TSG10 81.35 0.98 5 2.00 0.58 28.99

A- kinase anchor protein 3 A AP3 94.67 0.99 3 2.00 0.82 40.80

A lpha-1 -antitrypsin A1 AT 46.69 0.98 2 2.00 0.38 18.87

Ankyrin repeat domain- containing protein 36C AN36C 199.61 0.98 5 2.00 0.36 18.12

Ankyrin repeat domain- containing protein 62 ANR62 106.36 0.98 3 2.00 0.38 19.12

Basic helix-loop-helix

transcription factor

scleraxis sex 21.56 0.85 2 2.00 0.75 37.67

Bone morphogenetic

protein 15 BMP 15 45.01 0.97 2 2.00 0.66 32.81

Bromodomain-containing

protein 1 BRD1 1 19.43 0.98 3 2.00 0.72 36.04

Chromo s ome-as s ociated Q2KQ7

protein E 2 85.91 0.91 2 2.00 0.50 25.24

Cylicin-2 CYLC2 39.04 0.75 2 2.00 0.45 22.49

Cytosine-specific

methyltransferase F5GX68 183.25 0.99 4 2.00 0.33 16.75

Cytosoltc acyi coenzyme A F5GWE

thioester hydrolase 2 24.79 0.97 3 2.00 0.81 40.70

DNA topoisomerase 1 TOPI 90.65 1.00 3 2.00 0.81 40.37

G patch domain-containing

protein Ϊ GPTC1 103.26 0.96 3 2.00 0.54 27.22 Total

Protein Total Peptides

Weight Group {Paris % rel

Sequence Name Gene ID fkDa) Probability Guidelines) Intensity stdev stdev

Gamma-aminobutyric acid

receptor subunit delta GBRD 50.66 0.82 2 2.00 0.25 12.69

G-protein coupled receptor

98 GPR98 692.62 0.93 2 2.00 0.42 21.27

High affinity cAMP- specific and ΓΒΜΧ- insensitive 3',5 ^, -cyclic

phosphodiesterase 8B PDE8B 98.90 0.98 2 2.00 0.54 26.98

High affinity cGMP- specific 3',5'-cyclic

phosphodiesterase 9A PDE9A 68.43 0.95 2 2.00 0.49 24.45

HLA-DP protein Q30058 29.10 0.88 2 2.00 1.53 76.73

Inactive tyrosine-protein

kinase 7 PTK7 1 18.30 0.95 2 2.00 1.42 71.14

Integrator complex subunit

12 INT12 48.76 1.00 3 2.00 0,25 12.53

Interactor protein for

cytohesin exchange factors

1 ICEFl 48.94 0.98 4 2.00 0.07 3.37

Interleukin-l receptor-like

2 ILRL2 65.34 0.82 2 2.00 0.51 25.68

Kinesin-like protein IF27 KIF27 160.17 0.94 2 2.00 0.32 15.92

Leucine-rich repeat- containing protein 59 LRC59 34.89 0.96 2 2.00 0.78 38.95

Lysine-specific

demethylase 4C KDM4C 119.89 0.96 3 2,00 0.35 17.28

Msx2-interacting protein MINT 401.99 1.00 8 2.00 0.85 42.60

Neuropathy target esterase PLPL6 149.88 0.94 3 2.00 0.34 16.97

Phosphatidylinositol N- acetylglucosaminyltransfera

se subunit H PIGH 21.05 0.95 2 2.00 0.63 31.61

Phosphoryiated CTD- interacting factor 1 PCIFl 80.60 0.94 3 2.00 0.41 20.65

PML-RARA-regulated

adapter molecule 1 PRAM 79.18 0.95 4 2.00 1.01 50.59

POU domain, class A,

transcription factor 3 P04F3 37.01 0.84 2 2.00 0.31 15.44

POZ-, AT hook-, and zinc

finger-containing protein 1 PATZ1 73.99 0.87 3 2.00 0.89 44.66

Probable G-protein coupled

receptor 101 GP101 56.66 0.96 2 2.00 1.15 57.38

Probable G-protein coupled

receptor 157 GP157 36.58 0.96 2 2.00 0.48 23.82

Probable G-protein coupled

receptor 19 GPR19 47.64 0.98 2 2.00 0.50 24.92

Protein-methionine

sulfoxide oxidase MICAL3 MICA3 224.14 0.84 3 2.00 0,85 42.67

Rap guanine nucleotide

exchange factor 5 RPGF5 67.67 0.91 2 2.00 0.56 27.85 Total

Protein Total Peptides

Weight Group (Paris % rel

Seauence Name Gene ID (kDa) Probabilitv Guidelines) Intensity stdev stdev

Retinitis pigmentosa 1-like

1 protein RP1 L1 261.03 1.00 3 2.00 0.60 30.10

Ribosomal RNA processing

protein 1 homolog A RRP1 52.79 0.75 2 2.00 1.07 53.67

RNA-binding protein 28 RBM28 85.67 0.98 4 2.00 0.51 25,50

Serine/threonine-protein

kinase ATR ATR 301.15 0.99 4 2.00 0.34 17.16

Serologically defined colon

cancer antigen 8 SDCG8 82.61 0.99 4 2.00 0.74 36.94

SH3 and PX domain- containing protein 2A SPD2A 125.19 0.99 4 2.00 0.75 37.33

Signal recognition particle

68 kDa protein SRP68 70.67 1.00 2 2.00 1.02 50.88

Small subunit processome

component 20 homoiog UTP20 318.16 0.87 2 2.00 0.23 1 1.40

Structural maintenance of

chromosomes flexible

hinge domain-containing

protein 1 SMHD1 226.21 0.84 2 2.00 0.77 38.42

TATA-bindmg protein- associated factor 2N RBP56 61.78 0.90 3 2.00 0.45 22.62

Tenascin-X TENX 464.02 1.00 5 2.00 0.82 40.96

Tumor necrosis factor

receptor superfamily

member 8 TNR8 63.69 0.96 3 2.00 0.41 20.68

U4 U6.U5 small nuclear

ribonucleoprotein 27 kDa

protein SNR27 18.83 0.92 2 2.00 0.33 16.54

Unconventional myosin- VI MY06 149.58 0.86 3 2.00 0.86 43.26

Xanthine

d ehy drogenase/ oxid ase XDH 146.31 0.98 3 2.00 0.85 42.44

Zinc finger protein 221 ZN221 71.1 1 0.83 2 2.00 0.66 33.12

Zinc finger protein 300 ZN300 68.68 0.96 3 2.00 0.65 32.49

Zinc finger protein 426 ZN426 63.05 0.88 2 2.00 0.29 14.43

Zinc finger protein 445 ZN445 1 18.87 0.74 2 2.00 0.40 19.78

1 -phosphatidylinositol 4,5- bisphosphate

phosphodiesterase eta-2 PLCH2 154.55 0.96 3 2.00 0.67 33.46

Calcium-dependent

secretion activator 1 CAPS1 152.67 0.97 3 2.00 0.63 31.54

DDB1- and CUL4- associated factor 15 DCA15 66.40 0.97 2 2.00 0.30 14.90

Death domain-associated

protein 6 DAXX 81 .30 0.93 3 2.00 0.39 19.65

DNA excision repair

protein ERCC-6 ERCC6 168.29 0.94 2 2.00 0.59 29.77

DNA-binding protein

RFX6 RFX6 102.38 0.93 2 2.00 1.09 54.41

Ectopic P granules protein

5 homolog EPG5 292.27 0.72 2 2.00 0.32 16.16 Total

Protein Total Peptides

Weight GroHD ( Par is % rel

Sequence Name Gene ID fkDa Probability Guidelines) Intensity stdev stdev

ERBB receptor feedback

inhibitor 1 ERRFI 50.51 0.97 2 2.00 0.22 1 1.21

Inactive ubiquitin carboxyl- terminal hydrolase 54 UBP54 187.25 0.93 2 2.00 0.37 1 8.51

Insulinoma-associated

protein 1 INSM1 52.87 0.88 3 2.00 0.27 13.27

Leucine-rich repeat- containing protein 16B LR16B 150.12 0.98 3 2.00 0.72 35.88

MMRN

Multimerin-l 1 138.01 0.98 3 2.00 0.72 36.28

Noxl NAD(P)H-oxidase Q8TCT

subunit 3 58.91 0.91 2 2.00 0.50 25.01

Probable E3 ubiquitin- protein ligase MYCBP2 MYCB2 509.74 0.99 3 2.00 0.80 40.20

Ras-responsive element- binding protein 1 RREB1 181.29 0.96 3 2.00 1.00 49.91

Ryanodine receptor 3 RYR3 551.67 1.00 7 2.00 0.33 16.45

Solute carrier family 22

member 14 S22 66.62 0.97 3 2.00 0.57 28.35

Spindle assembly abnormal

protein 6 homolog SAS6 74.33 0.87 2 2.00 0.82 40.88

Q4KN0

Taste receptor type 2 4 35.87 1.00 2 2.00 0.91 45.64

TBC1 domain family

member 9 TBCD9 143.12 0.89 3 2.00 0.29 14.32

TTC6 protein Q3SY86 68.47 0.96 2 2.00 0.95 47.47

Ubiquitin carboxyl-terminal

hydrolase 34 UBP34 403.95 1.00 6 2.00 0.63 31.69

Unconventional myosm- XVIIIa MYI 8A 232.95 0.84 3 2.00 0.3 1 15.28

Zinc finger protein 468 ZN468 60.52 0.95 3 2.00 0.76 37.87

Serine/threonine-protein

kinase 10 STK10 1 12.05 0.98 2 2.00 0.26 13.07

Protein kinase C epsilon

type K.PCE 83.60 0 ^' .99 2 1.00 0.42 42.15

Serine/threonine-proteitt

kinase OSR1 OXSR1 57.97 0.98 2 1.00 0.54 53.54

Tyros ine-prote in

phosphatase non-receptor

type 11 ΡΤΝΠ 68.38 0.97 3 1.00 0.43 43.13

ATP-dependent RNA

helicase DD 18 DDX18 75.34 1.00 3 1.00 0.42 41.51

Coiled-coil domain- containing protein 86 CCD86 40.19 0.95 2 1.00 0.42 41.50

Mito gen-activated protein

kinase kinase kinase 9 M3K9 121.80 0.84 2 LOO 0.24 23.65

Q49AL

OLIG2 protein 3 32.39 0.81 2 1.00 0.49 48.38

Probable ATP-dependent

RNA helicase DDX60-like DDX6L 197.53 0.91 3 1.00 0.47 47.12 Total

Protein Total Peptides

Weight Group {Paris % rel

Sequence Name Gene ID fkDa) Probability Guidelines Intensity stdev stdev

Probable ubiquitin

carboxyl-terminal

hydrolase FAF-X USP9X 292.08 0.92 3 1.00 0.23 22.54

Protein TANC1 TANC1 202.07 0.95 3 1.00 0.37 36.56

Versican core protein CSPG2 372.57 0.74 2 LOO 0.53 52.44

Dedicator of cytokinesis

protein 5 DOCKS 215.15 0.98 3 1.00 0.28 27.76

2-5A-dependent

ribonuc lease RN5A 83.46 0.97 2 1.00 0.54 54.20

6-phosphofructo-2- kinase/fructose-2,6- bisphosphatase 2 F262 58.42 0.99 3 1.00 0.44 43.61

A disintegrin and

metal proteinase with

thrombospondin motifs 2 ATS2 134.65 0.94 2 1.00 0.20 20.05

AF4/FMR2 family member

3 AFF3 133.38 0.99 5 1.00 0.23 23.46

Aprataxin APTX 40.70 0.92 2 1.00 0.53 53.22

ATP-binding cassette subfamily A member 7 ABCA7 234.18 0.77 2 1.00 0.23 23.25

ATP-binding cassette subfamily A member 9 ABCA9 184.22 0.85 2 1.00 0.32 31.50

Basic helix-loop-helix

domain-containing protein

KIAA2018 K2018 241.51 0.98 3 1.00 0.77 77.25

BRCA2 and CDKN1A- interacting protein BCCIP 35.94 0.89 2 1.00 0.61 61.21

Calcium-activated chloride

channel regulator 1 CLCA1 100.15 0.89 3 1.00 0.42 42.05

Cap-specific mRNA

(nucIeoside-2 ^! -0-)- methyltransferase 1 MTR1 95.24 0.92 3 1.00 0.46 45.63

CDS antigen-like CD5L 38.04 0.98 2 3 .00 0.32 32.20

Centriolar coiled-coil

protein of 1 10 kDa CP1 10 1 13.33 0.99 3 1.00 0.70 70.04

Coiled-coil domain- containing protein 106 CC106 31 .99 1.00 3 1 ,00 0.36 36.19

Coiled-coil domain- containing protein 60 CCD60 63.03 0.92 2 1.00 0.50 49.43

Collagen alpha-l(XV)

chain COFA1 141.61 0.93 2 1.00 0.65 65.12

Complement factor 1 CFAI 65.69 1.00 4 3 .00 0.31 30.57

Cycl in-dependent kinase- like 5 CDKL5 1 15.45 1.00 5 1.00 0.50 49.63

DCC-interacting protein

13-beta DP13B 74.43 1 .00 2 1.00 0, 17 17.26

Dehydrogenase/reductase

SDR family member 4 DHRS4 29.50 0.95 2 1.00 0.34 34.37

DEN domain-containing

protein 2C DEN2C 106.78 0.79 2 1.00 0.55 54.97 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa) Probability Guidelines Intensity stdev stdev

DNA repair protein

XRCC1 XRCC I 69.42 0.76 3 1.00 0.66 65.71

DN A -binding protein

RFX8 RFX8 66.21 0.96 3 1.00 0.48 47.91

Docking protein 2 DO 2 45.33 0.90 2 1.00 0.29 29.1 1

Dynein heavy chain 3,

axonemal DYH3 470.45 0.97 2 1.00 0.74 73.96

E3 ubiquitin-protein ligase

UBR3 UBR3 212.28 1.00 4 1.00 0.33 32.48

Ectoderm-neural cortex

protein 2 ENC2 65.86 0.71 2 1.00 0.32 32.22

EF-hand domain-containing

protein D2 EFHD2 26.66 0.82 2 1.00 0.32 32.36

A6H8Y

EML4 protein 6 102.38 0.99 3 1.00 0.16 16.42

ERC protein 2 ERC2 1 10.47 0.98 2 1.00 0.08 7.85

Ermin ERMIN 32.74 0.78 2 1.00 0.47 46.50

Fanconi anemia group M FANC

protein M 232.03 1.00 4 1.00 0.19 19.09

Fatty-acid amide hydrolase

2 FAAH2 58.25 0.93 2 1.00 0.21 21.43

Frizzled homolog 9 Q8TAN

(Drosophila) 2 64.46 0.98 2 1.00 0.30 30.01

G protein-activated inward

rectifier potassium channel

4 IRKS 47.62 0.91 2 1.00 0.50 50.06

Q6B6N

Galphai2 protein 3 41.50 0.86 2 LOO 0.13 12.65

GH3 domain-containing

protein GFIDC 57.47 1.00 2 1.00 0.54 54.16

Golgi apparatus protein 1 GSLG1 134.45 0.92 3 1.00 0.21 20.72

Golgin subfamily A

member 8J GOGSJ 65.61 0.93 3 1.00 0.17 16.70

Granzyme K GRAK 28.85 0.86 2 1.00 0.31 30.75

GTP-binding protein 2 GTPB2 65.71 0.97 2 1.00 0.67 66.46

Guanylate-binding protein

3 GBP3 68.05 0.99 2 1.00 0.47 46.82

High mobility group

protein B3 HMGB3 22.95 0.98 2 1.00 0.49 48.49

Q9HA1

Histone H2A 1 29.86 0.84 2 1.00 0.26 26.04

Histone-lysine N- methyltransferase MLL3 MLL3 541.01 1.00 6 1.00 0.28 28.03

Inhibitor of nuclear factor

kappa-B kinase subunit

beta IKKB 86.49 0.79 2 1.00 0.35 34.61

Inositol 1 ,4,5-trisphosphate

receptor type 3 ITPR3 303.89 0.70 2 1.00 0.70 70.13

Insulin receptor substrate 2 IRS2 137.23 0.92 3 1 .00 0.24 24.25

Insulin-degrading enzyme IDE 1 17.88 0.90 2 1.00 0.58 57.87 Total

Protein Total Peptides

Weight Group (Paris % rei

Sequence Name Gene ID (kDa) Probability Guidelines) Intensity stdev stdev

Inverted formin-2 INF2 135.52 0.99 3 1.00 0.52 51.87

IQ domain-containing

protein E IQCE 77.23 0.88 2 1.00 0.18 17.79 elch-iike protein 32 70.30 0.91 2 1.00 0.23 23.30

Kelch-!ike protein 7 LHL7 65.93 0.91 3 1.00 0.32 31 .72

Keratin 77 Q0IIN 1 61.75 1.00 3 1.00 0.50 50.28

Kinesin-like protein

KIF18A ΚΓ18Α 102.20 0.83 2 1.00 0.48 47.49

Low-density lipoprotein

receptor-related protein 6 LRP6 180.30 0.73 2 1.00 0.19 18.60

Ly mphoid-restricted

membrane protein LRMP 62.07 0.88 2 1.00 0.32 32.40

Lysine-specific

demethylase 3B KDM3B 191 .44 0.91 3 1.00 0.29 28.47

Microtubule-associated

protein 2 MAP2 199.39 0.96 4 1.00 0.31 31 .17

Mucin-5B MUC5B 595.94 1.00 4 1.00 0.70 69.43

Myotubu!arin-related MTMR

protein 10 A 88.20 0.79 2 1.00 0.12 12.30

Nance-Horan syndrome

protein NHS 176.57 0.93 4 1.00 0.33 32.45

NF-Xl-type zinc finger

protein NFXL1 NFXL1 101.25 0.87 2 1.00 0.42 43 .52

Nuclear auto antigen Sp-100 SP100 100.34 0.99 4 1.00 0.70 70.19

Nuclear distribution protein

nudE-Iike 1 NDEL1 38.33 0.95 3 1 .00 0.52 52.21

Nuclear receptor-binding

protein 2 NRBP2 57.75 0.92 2 1.00 0.43 43.16

Parvalbumin alpha PRVA 12.03 0.89 2 1.00 0.70 69.82

PHD finger protein 10 PHF 10 56.00 0.92 4 1.00 0.35 34.70

Phosphofurin acidic cluster

sorting protein 2 PACS2 97.62 0.71 2 1.00 0.43 43.34

Piwi-like protein 4 PIWL4 96.51 0.87 2 1.00 0.65 64.58

Placenta apolipoprotein

B48 receptor type 2 Q9NS 13 1 13.35 0.90 2 1.00 0.45 45.35

Potassium voltage-gated

channel subfamily KQT A6PVT

member 5 6 104.46 0.98 2 1.00 0.26 25.77

PR domain zinc finger

protein 10 PRD10 130.03 0.80 2 1.00 0.16 16.09

Probable E3 ubiquitin- protein ligase TRIP 12 TRIPC 220.28 0.86 3 1.00 0.28 28.06

Probable helicase senataxin SETX 302.67 0.95 4 1.00 0.61 61.26

Probable RNA-binding

protein 20 RBM20 134.26 0.89 3 1 .00 0.14 14.04

Prolow-density lipoprotein

receptor-related protein 1 LRP1 504.26 1.00 7 1 .00 0.17 17.33

Proprotein convertase

subtil isin kexin type 5 PCSK5 206.78 0.95 3 1 .00 0.41 41.22 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID OkDa) Probability Guidelines) Intensity stdev stdev

Proteasome activator

complex subunit 4 PSME4 21 1.18 0.95 3 1.00 0.18 18.24

Proteasome-associated

protein ECM29 homolog ECM29 204.14 0.88 2 1.00 0.16 16.14

Protein 4.1 41 96.94 0.99 3 1.00 0.53 53.21

Protein CBFA2T1 MTG8 67.51 0.71 2 1 ,00 0.27 27.20

Protein FAM1 1 1B F1 1 1B 84.60 0.91 2 1.00 0.57 57.04

Protein FA 161B F16IB 73.58 0.77 3 1.00 0.20 20,00

Protein FAM179A F179A 1 1 1.07 0.95 3 1 .00 0.31 31.18

Protein FAM47B FA47B 73.88 0.89 2 1.00 0.45 44.59

Protein kinase, cAMP- dependent regulatory, type Q6DHZ

II, beta 2 46.30 0.96 3 1.00 0.32 32.00

Protein NDRG4 NDRG4 38.42 0.97 2 1.00 0.15 14.56

Protocadherin alpha-1 1 PCDAB 103.22 0.96 3 1.00 0,39 38.96

RalBPl -associated Eps

domain-containing protein

1 REPS1 86.59 0.73 2 1.00 0.28 27.56

Ras GTPase-activating

protein-binding protein 2 G3BP2 54.07 0.89 3 1.00 0.41 40.64

Ras GTPase-activating-like

protein IQGAP3 IQGA3 184.57 0.77 2 1.00 0.28 28.1 1

Ras guanyl -re leasing

protein 3 GRP3 78.26 0.89 2 1.00 0.32 31.57

Ras-specific guanine

nucleotide-releasing factor

2 RGRF2 140.66 0.85 2 1 ,00 0.22 22.31

RBl-inducible coiled-coil

protein 1 RBCC1 182.96 1.00 4 1.00 0, 14 13.80

Receptor-type tyrosine- protein phosphatase 0 PTPRO 138.24 0.83 2 1.00 0.31 31.18

Retinoblastoma-associated

protein RB 106.07 0.77 2 1.00 0.24 23.95

R o guanine nucleotide ARHG

exchange factor 16 G 80.04 1.00 2 1.00 0.15 15.12

Ribosomal protein L29 Q5T1D

(RPL29) pseudogene 1 17.34 0.84 2 1.00 0.36 36.39

RNA polymerase II- associated protein 1 RPAP1 152.64 0.75 2 1.00 0.28 28.37

RNA-binding protein 25 RBM25 100.11 0.82 2 3 .00 0.55 54.64

RNA-binding protein with

serine-rich domain 1 RNPS 1 34.17 0.80 2 1.00 0.08 7.54

Secitrin-2 PTTG2 22.27 0.95 2 3 .00 0.33 33.23

Sepiapterin reductase SPRE 28.01 0.96 2 1 .00 0.73 72,90

Serine/threonine-protein

kinase DCL 1 DCL 1 82.15 0.87 2 1.00 0.51 51 ,31

Serine/threonine-protein

phosphatase 2 A 55 kDa

regulatory subunit B delta

isoform 2ABD 51.99 0.89 3 3.00 0.19 19.41 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID fkDa) Probability Guidelines') Intensity stdev stdev

Serine/threonine-protein

phosphatase 2B catalytic

subunit gamma isoform PP2BC 58.07 0.92 2 1.00 0.33 32.82

Short stature homeobox

protein 2 SHOX2 34.91 1.00 2 1.00 0.07 7.07

Sickle tail protein homolog SKT 213.96 1.00 2 1.00 0,56 56,35

Signal peptide peptidase- like 2C SPP2C 74.44 0.86 2 1.00 0.42 42.39

Signal-induced

proliferation-associated 1- like protein 1 SI1L1 199.89 0.74 2 1.00 0.50 50.15

Signal-induced

proliferation-associated

protein 1 SIPA1 1 12.06 0.88 3 1.00 0.43 42.61

SLAIN motif-containing

protein 2 SLAI2 62.49 0.99 2 1.00 0.37 36.57

S-methylmethionine—

homocysteine S- methyitransferase BHMT2 BHMT2 40.31 1.00 2 1.00 0.32 31.83

Q86XC

Sorting nexin 13 4 1 10.90 1.00 7 1.00 0.78 78.18

Sorting nexin-6 SNX6 46.60 0.88 2 1.00 0.21 21.31

Sterol regulatory element- binding protein 1 SRBPl 121.58 0.77 3 1.00 0.21 21.09

Syntaxin-2 STX2 33.30 0.78 2 1.00 0.56 55.88

T-complex protein 1

subunit delta TCPD 57.87 1.00 2 1.00 0.42 41.55

Teashirt homolog 3 TSH3 118.47 0.88 2 1.00 0.33 32.86

Testis specific basic protein Q13647 63.26 0.91 2 1.00 0.65 64.70

THAP domain-containing

protein 9 THAP9 103.33 1.00 4 1.00 0.51 51.19

Thrombospondin type-1

domain-containing protein

4 THSD4 1 12.36 1.00 3 1.00 0.41 40.56

Thyroid adenoma- associated protein THADA 219.45 0.89 4 1.00 0.68 67.91

T-lymphoma invasion and

metastasis-inducing protein

2 TIAM2 189.97 0.97 2 1.00 0.47 47.26

TNF receptor-associated

factor 5 TR.AJF5 64.35 0.99 3 1.00 0.49 48,43

Transcription factor 20 TCF20 21 1.62 1.00 4 1.00 0.16 16.40

Transcription factor

HIVEP3 ZEP3 259.29 0.93 3 1 .00 0.44 44.28

Transcription initiation

factor TFIID subunit 1 -like TAF1L 207, 15 1.00 4 1.00 0.37 36,55

Transcription intermediary

factor 1 -alpha TIF1A 1 16.74 0.71 2 1.00 0.26 26,41

Transforming growth factor

beta receptor type 3 TGBR3 93.42 0.88 2 1.00 0.68 67,66 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa) Probability Guidelines) Intensity stdev stdev

Translation initiation factor

IF-3, mitochondrial IF3M 31.69 0.95 3 1.00 0.53 52.64

Translocation protein

SEC62 SEC62 45.82 0.96 3 1.00 0.16 15.83

Transmembrane and coiled- coil domain-containing

protein 7 TMC07 120.65 0.99 4 1.00 1.01 101.00

Tripartite motif-containing

protein 69 TRI69 57.36 0.93 2 1.00 0.35 14.76

Tudor domain-containing

protein 3 TDRD3 73.12 0.80 2 1.00 0.24 23.74

Tyrosine-protein kinase

transmembrane receptor

ROR2 ROR2 104.67 0.99 3 1.00 0.13 33.34

Wiskott-Aldrich syndrome

protein family member 2 WASF2 54.23 0.88 2 1.00 0.47 47.29

Zinc finger and BTB

domain -containing protein

41 ZBT41 105.1 1 0.95 3 1.00 0.41 41.25

Zinc finger and SCAN

domain-containing protein

22 ZSC22 54.51 0.95 2 1.00 0.22 21.71

Zinc finger protein 319 ZN31 65.49 0.85 2 1.00 0.18 17.58

Zinc finger protein 518B Z518B 1 19.44 0.90 3 1.00 0.58 58.37

Zinc finger protein 519 ZN519 62.96 0.97 3 1.00 0.40 40.06

Zinc finger protein 618 ZN618 104.87 0.73 2 1.00 0.49 48.58

Zinc finger protein 678 ZN678 61.35 0.95 2 1.00 0.32 31.83

Zinc finger protein 699 ZN699 73.89 0.79 2 1.00 0.28 28.05

Zinc finger protein 805 ZN805 71.08 0.93 2 1.00 0.85 85.32

Zinc finger protein 813 ZN 13 71.66 0.92 2 LOO 0.97 96.51

Zinc finger Ran-binding

domain-containing protein

2 ZRAB2 37.36 0.94 2 LOO 0.29 28.83

Coiied-coil and C2 domain- containing protein 2A C2D2A 186.05 0.89 2 LOO 0.46 46.40

Is o leucine— tRNA ligase,

mitochondrial SYIM 1 13.70 0.99 3 LOO 1.01 100.59

Protocadherin alpha-9 PCDA9 102.32 0.76 2 LOO 0.37 37.20

UDP-glucose:glycoprotein

glucosyltransferase 1 UGGG1 177.06 0.85 2 1 .00 0.20 19.62

Zinc finger protein 541 ZN541 147.70 0.89 2 1.00 0.35 34.56

Alpha- 1 ,3-mannosyl- glycoprotein 4-beta-N- acetylglucosaminyltransfera

se A MGT4A 61.49 0.99 3 LOO 0.22 21.93

AN 1 -type zinc finger

protein 4 ZFAN4 80.29 0.97 3 LOO 0.68 68.1 1

Argmme/serine-rich protein

PNISR PNISR 92.50 0.73 2 LOO 0.32 32.29 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa) Probability Guidelines) Intensity stdev stdev

CTTNBP2 N-terminal-like

protein CT2NL 70.10 0.97 2 1.00 0.51 51.29

Cytoplasmic dynein 2

heavy chain 1 DYHC2 492.29 1.00 7 1.00 0.49 49.15

Dual specificity protein

kinase TTK TTK 96.99 0.79 2 1.00 0.60 59.51

Dynein heavy chain 10,

axonemal DYH10 514.49 1.00 6 1.00 0.43 43.43

Dynein heavy chain 2,

axonemal DYH2 507.36 0.98 3 1.00 0.44 43.66

E3 ubiquitin-protein ligase

HUWE1 HUWE1 481.57 1.00 7 1.00 0.54 54.31

Epidermal growth factor

receptor substrate 15-like 1 EP15R 94.18 0.82 2 1.00 0.19 19.36

FH1/FH2 domain- containing protein 3 FHOD3 158.50 0.94 3 1.00 0.81 80.61

Filamin-A-interacting

protein 1 FLIP1 138.01 0.97 3 1.00 0.34 34.25

FYVE, RhoGEF and PH

domain-containing protein

4 FGD4 86.55 0.83 2 1.00 0.37 37.43

Gamma-aminobutyric acid

receptor subunit alpha-5 GBRA5 52.09 0.91 3 1.00 0.48 48,03

Huntingtin HD 347.36 0.84 3 1.00 0.40 40.48

Huntingtin-associated

protein 1 HAP1 75.44 0.95 2 1.00 0.45 45.01

Inhibitor of nuclear factor

kappa-B kinase subunit

alpha IKKA 84.57 0.99 3 1.00 0.81 81.46

Interleukin-33 IL33 30.72 0.84 2 1.00 0.79 79.03

KRAB domain-containing

protein ZNF747 ZN747 20.58 0.91 2 1.00 0.1 1 10.86

Leukocyte elastase inhibitor 42.70 0.85 3 1.00 0.14 14.38

Lysine-specific KDM5

demethylase 5 A A 191.95 0.99 5 1.00 0.39 38.50

Matrilin-3 MATN3 52.76 0.89 2 1.00 0.15 15.17

Mitofusin-1 MFN1 84.03 0.98 3 1.00 0.28 28.19

Myotubularin-related MTMR

protein 5 5 208.17 0.93 3 1.00 0.31 30.71

NF-kappa-B essential

modulator NEMO 48.15 0.92 2 1.00 0.36 36.07

NK-tumor recognition

protein NKTR 165.56 1.00 5 1.00 0.65 64.90

NLR family CARD

domain-containing protein

4 NLRC4 1 16.07 0.93 3 1.00 0.32 31.92

Primary ciliary dyskinesia

protein 1 PCDP1 96.81 0.95 3 1.00 0.46 45.98 Total

Protein Total Peptides

Weieht Group (Paris % rel

Sequence Name Gene ID ( ⁾ a) Probability Guidelines) Intensity stdev stdev

Probable tRNA

threonylcarbamoyladenosin

e biosynthesis protein

OSGEP OSGEP 3639 0.97 2 1.00 0.60 60.37

Protem Wnt-lOb WN10B 42.95 0.98 3 LOO 0.32 31.81

Ras-reiated and estrogen- regulated growth inhibitor RERG 22.58 0.98 2 1.00 0.29 28.70

Rho guanine nucleotide ARHG

exchange factor 26 Q 97.27 0.97 2 1.00 0.35 35.32

Ribosome-recycling factor,

mitochondrial RRFM 29.24 0.97 2 1.00 0.33 32.61

Testican-3 TICN3 49.38 0.94 2 LOO 0.52 51.68 tRNA wybutosine- synthesizing protein 1

homolog B TYW1B 76.88 0.96 2 LOO 0.25 25.27

Tubulin-specific chaperone

D TBCD 132.50 0.88 ₂ LOO 0.68 68.15

Up-regulator of cell

proliferation URGCP 104.90 0.82 2 LOO 0.23 23.24

V-type proton ATPase

catalytic subunit A VATA 68.24 0.94 2 LOO 0.51 50.81

Zinc finger protein 106

homolog ZF106 208.74 0.94 2 LOO 0.28 27.92

Zinc finger protein 592 ZN592 137.42 0.84 3 LOO 0.10 10.04

17-beta-hydroxysteroid

dehydrogenase type 6 H17B6 35.92 0.77 2 3 .00 0.40 39.71

1 -phosphatidylinositol 4,5- b is phosphate

phosphodiesterase beta-1 PLCB 1 138.46 0.79 3 LOO 0.63 63.30

1 -phosphatidylinositol 4,5- bisphosphate

phosphodiesterase epsilon- 1 PLCE1 258.53 0.74 2 LOO 0.40 39.62

5 -hydroxytryptamine F5GWE

receptor 2A 8 43.89 0.88 2 LOO 0.52 51.56

5 '-nucleotidase domain- containing protein 4 NT5D4 48.90 0.71 2 LOO 0.38 37.56

5-oxoproiinase (ATP- hydrolysing) A7E261 137.32 1.00 3 LOO 0.22 22.35

60S ribosomal protein L28 RL28 15.72 0.85 2 1.00 0.24 24.02

60S ribosomal protein L6 RL6 32.69 0.92 2 1.00 0.42 42.38

Acety lch oi inesterase C9JGQ9 65.30 0.96 2 LOO 0.13 12.57

Acidic repeat-containing

protein ACRC 76.05 0.98 2 LOO 0.45 44.60

ADAMTS-like protein 2 ATL2 104.53 0.75 2 LOO 0.50 50.28

Adenomatous polyposis

coli protein 2 APC2 243.78 1.00 4 LOO 0.75 75.43

A-kinase anchor protein 1 ,

mitochondrial AKAP1 97.26 0.83 2 1.00 0.45 44,57

A-kinase anchor protein 4 AKAP4 94.40 0.92 2 LOO 0.32 31.64 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID ikDa) Probability Guidelines) Intensity stdev stdev

Alpha N-terminai protein

methyltransferase 1 A NTM1A 25.35 0.94 3 1.00 0.52 52.12

Alpha-protein kinase 2 J .UPK.2 236.85 0.96 3 LOO 0.31 30.59

Amiloride-sensitive cation

channel 4 ACCN4 70.04 0.92 2 1.00 0.49 49.47

Aminopeptidase Q AMPQ 1 13.19 0.82 2 1 .00 0.21 20.97

Q5W02

Ankyrin repeat domain 3 OA 6 165.75 0.99 4 1.00 0.68 67.81

Ankyrin repeat domain- containing protein 20B AN20B 93.83 0.93 2 1.00 0.35 34.84

Ankyrin repeat domain- containing protein 36A AN36A 217.31 0.99 3 1 .00 0.16 16.24

Ankyrin repeat domain- containing protein 53 ANR53 59.49 0.99 4 1 .00 0.50 49.86

Armadillo repeat- containing X-linked protein

2 ARMX2 65.62 0.95 2 1 .00 0.74 73.95

ATPase family AAA

domain-containing protein

2B ATD2B 164.79 0.98 4 1.00 0.41 41.02

Autism susceptibility gene

2 protein AUTS2 138.88 0.89 2 1 .00 0.27 27.42

Band 4, 1 -like protein 4A E41 LA 78.99 0.82 2 1 .00 0.66 65.88

BEN domain-containing

protein 5 BENDS 48.13 0.97 2 1 .00 0.47 46.84

BTB/POZ domain- G3V3T

containing protein 7 2 54.31 0.80 2 1.00 0.15 14.82

Calcyphosin-like protein CAPSL 24.20 0.78 2 1.00 0.18 18.01

Calsyntenin-2 CSTN2 106.92 0.97 3 1.00 0.29 29.37

G5E9U

Cdc42-interacting protein 4 1 67.67 0.98 2 1.00 0.68 67.50 cDNA PSEC0102 fis, clone

NT2 P2003127, highly B3KQS

similar to Prominin-1 1 96.1 1 1.00 3 1.00 0.72 72.24

CDV3 homolog (Mouse), D3DND

isofonn CRA a 1 28.56 0.99 3 1.00 0.17 17.13

Cell growth-inhibiting

protein 34 Q08ES8 20.06 0.99 2 1.00 0.64 64.29

Centrosomal protein of 104

kDa CE104 104.36 0.92 2 1.00 0.37 36.74

Centrosomal protein of 95 HOYJW

kDa 6 87.93 1.00 4 1.00 0.71 71 .37

Cholesterol 24-hydroxyiase CP46A 56.77 0.74 2 1.00 0.59 58.73

Choline kinase alpha CHKA 52.20 0.73 2 1.00 0.19 19.48

Chromosome 21 open

reading frame 63, isoform A6ND5

CRA c 8 49.08 0.90 2 1.00 0.3 1 31 .40

Clusterin-associated protein

1 CLUA1 48.08 0.87 2 3 .00 0.49 49.06 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID fkDa Probability Guidelines) Intensity stdev stdev

Coiled-coil domain- containing protein 146 CC146 1 12.72 0.98 2 1.00 0.61 60.54

Coiled-coil domain- containing protein 1 52 CC152 29.94 0.90 2 1.00 0.10 10.00

Coiled-coil domain- containing protein 162 CC162 103.76 0.79 2 1.00 0.50 49.67

Coiled-coil domain- containing protein 36 CCD36 66.28 1.00 3 1.00 0.27 26.56

Coiled-coil domain- containing protein 7 CCD37 71.06 0.84 3 1.00 0.46 45.94

Coiled-coil domain- containing protein 57 CCD57 103.09 0.98 2 1.00 0.34 33.63

Coi!ed-coil domain- containing protein 73 CCD73 124.06 0.95 3 1.00 0.50 50.18

Coiled-coil domain- containing protein 91 CCD91 49.92 0.86 2 1.00 0,61 60.63

Coiled-coil domain- containing protein lobo

homolog CC135 103.41 0.99 3 1.00 0.46 45.66

CXXC-type zinc finger

protein 4 CXXC4 20.95 0.73 2 1.00 0.30 30.07

Cysteine/serine-rich nuclear

protein 1 CSRN1 63.45 0.80 2 1.00 0.33 32.67

Cysteine-rich protein 2- binding protein CSR2B 88,77 0.93 3 1.00 0.48 47.53

Cytohesin-4 CYH4 45.63 0.93 2 1.00 0.92 92.12

Cytoplasmic dynein 1

heavy chain 1 DYHC1 532.05 0.92 2 1.00 0.40 39.89

Cytoske 1 eton-associ ated

protein 2-Iike C P2L 83.52 1.00 3 1.00 0.60 59.95

Dermatan-sulfate epimerase DSE 109.69 0.79 2 1.00 0.20 19.52

Diacylglycerol kinase eta DG H 134.76 0.82 3 1.00 0.56 55.90

Disheveled-associated

activator of morphogenesis DAAM

2 2 123.40 0.83 2 1.00 0.56 55.75

Disks large-associated

protein 1 DLGP1 108.79 0.87 2 1.00 0.31 31.41

DmX-like protein 3 DMXL1 337.61 0.99 3 1.00 0.39 38.77

DNA polymerase theta Q96SE4 304.94 0.99 3 1.00 0.14 13.91

DNA repair endonuclease

XPF XPF 104.40 0.87 3 1.00 0.18 17.66

DNA topoisomerase 2-beta TOP2B 183.13 LOO 6 1.00 0.58 57.57

DNA-directed RNA

polymerase II subunit

RPB2 RPB2 133.79 0.80 3 1.00 0.12 1 1.58

DnaJ (Hsp40) homolog,

subfamily A, member 1 ,

isoform CRA b Q86TL9 37.00 0.98 2 1.00 0.23 23.03

Doublecortin domain- containing protein 1 DCDC1 39.82 0.94 2 1.00 0.58 58.46 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID fkDa) Probability Guidelines) Intensity stdev stdev

Double-strand-break repair

protein rad21 homoiog RAD21 71 ,63 0.95 2 1.00 0.22 22.01

Double-stranded RNA- specific adenosine

deaminase DSRAD 135.96 1.00 5 1.00 0.72 72.24

Dynamin-like 120 kDa

protein, mitochondrial OPA1 1 1 1.54 0.87 2 1.00 0.38 37.96

El A-binding protein p400 EP400 343.26 0.80 2 1 ,00 0.48 47.76

E3 ubiquitin-protein ligase

RBBP6 RBBP6 201.42 0.87 3 1.00 0.30 30.36

E3 ubiquitin-protein ligase

SH3RF1 SH3R1 93.05 0.92 2 1.00 0.12 11.76

E3 ubiquitin-protein ligase

TRIM37 TRI37 107.82 1.00 3 1.00 0.18 17.54

E3 ubiquitin-protein ligase

TRIM68 TPJ68 56.21 1.00 ₂ 1.00 0.21 21.31

Echinoderm microtubule- associated protein-like 5 EMAL5 219.27 0.97 4 1.00 0.21 20.83

Ectonucleotide

pyrophosphatase/phosphodi

esterase family member 2 ENPP2 98.91 0.83 2 1.00 0.68 67.97

Eukaryotic translation

initiation factor 3 subunit D EIF3D 63.91 0.91 2 1.00 0.44 43.85

Eukaryotic translation

initiation factor 3 subunit M EIF3M 42.46 0.93 2 1.00 0.46 45.89

Eukaryotic translation

initiation factor 4E

transporter 4ET 108.12 0.99 3 1.00 0.40 40.04

Exocyst complex

component 3 EXOC3 86.77 0.95 2 1.00 0.17 17.40

Extracellular sulfatase Sulf- 1 SULF1 100.94 0.90 3 1.00 0.50 50.30

Fanconi anemia group B

protein FANCB 97.65 0.81 2 1.00 0.83 82.69

Q4VBP

F-box protein 34 5 78.63 1.00 3 1.00 0.13 12.96

Fetal beta-MHC binding

factor Q14297 44.08 1.00 4 1.00 0.41 40.85

Fibri!lin-2 FBN2 314.54 0.82 2 LOO 0.72 72.42

Fibrous sheath-interacting

protein I FSIP1 66.06 0.84 2 1.00 0.14 14.17

G patch domain-containing

protein 2 GPTC2 58.89 1.00 2 1.00 0.52 51.61

G patch domain-containing

protein 4 GPTC4 50.33 0.98 3 1.00 0.47 47.01

GDP-L-fucose synthase FCL 35.85 0.83 2 1.00 0.60 60.48

GDP-mannose 4,6

dehydratase GMDS 41.91 0.96 3 1.00 0.32 31.83

General transcription factor

3C polypeptide 1 TF3C1 238.71 0.98 2 1.00 0.41 41.18 Total

Protein Total Peptides

Weight Group (Paris % rel

Se uence Name Gene ID OtDa) Probability Guidelines) Intensity stdev stdev

General transcription factor

3C polypeptide 5 TF3C5 59.52 0.88 2 1.00 0.29 29.25

Glutathione reductase,

mitochondrial GSHR 56,20 0.94 3 1.00 0.20 20.28

Golgin subfamily A

member 4 GOGA4 260.96 1.00 5 1.00 0.38 38.28

GRIP 1 -associated protein 1 GRAP1 95.91 0.87 2 1.00 0.34 33.80

GTP-binding protein RAD RAD 33.21 0.88 2 1.00 0.16 16.27

Heat shock protein beta-1 HSPB1 22.75 1.00 2 1.00 0.68 67.55

Helicase POLQ-like HELQ 124.03 0.87 2 1.00 0.64 63.84

Heparan sulfate 2-0- sulfotransferase 1 HS2ST 41 .84 0.98 2 1.00 0.44 44.49

Hepatocyte growth factor HGF 83.06 1.00 2 1.00 0.49 49.00

Heterogeneous nuclear

ribonucleoprotein H HNRH1 49.18 0.99 3 1.00 0.38 38.41

Histone deacetylase 9 HDAC9 1 1 1.21 0.82 2 1.00 0.24 24.02

Histone HI .3 H13 22.32 0.84 2 1.00 0.55 55.37

Histone-lysine N- methyltransferase EZH2 EZH2 85.29 0.85 2 1.00 0.37 37.45

Histone-lysine N- methyltransferase MLL4 MLL4 293.31 0.98 4 1.00 0.17 16.54

HMG domain-containing HMGX

protein 3 3 168.21 0.84 2 1.00 0.28 27.80

HSPC341 Q9P0I9 19.86 0.91 2 1.00 0.70 69.55

Hyaiuronan and

proteoglycan link protein 1 HPLN1 40.12 0.81 2 1.00 0.24 24.48

INO80 complex subunit B IN80B 38.59 1.00 3 1.00 0.92 91.92

Insulin-like growth factor- binding protein 2 IBP2 34.77 0.84 2 1.00 0.43 42.82

Insulin-like growth factor- binding protein 3 IBP3 31.64 0.91 3 1.00 0.76 75.66

Integrin alpha- 7 ITA7 128.85 0.91 2 1.00 0.42 42.25

Inter-alpha- trypsin inhibitor

heavy chain H5 ITIH5 104.49 0.87 2 1.00 0.33 32.51

Intercellular adhesion Q8TAM

molecule 5, telencephalin 9 97.08 1.00 3 1.00 0.78 78.45

Interferon-induced very

large GTPase 1 GVINI 278.85 0.81 2 1.00 0.27 26.53

Interleukin enhancer- binding factor 3 ILF3 95.26 0.84 3 1.00 0.42 41.85

Isocitrate dehydrogenase

[NADP], mitochondrial IDHP 50.86 0.83 2 LOO 0.22 21.68

Junctional protein

associated with coronary

artery disease JCAD 148.24 0.89 3 1.00 0.32 32,31

Kelch-like protein 8 LHL8 68.74 0.97 2 1.00 0.28 28.10

KH domain-containing,

RNA-binding, signal

transduction-associated

protein 1 HDR1 48.18 0.77 2 1.00 0.22 22.1 1 Total

Protein Total Peptides

Weight Group (Paris % rei

Sequence Name Gene ID fkDa) Probability Guidelines) Intensity stdev stdev

Kinesin-1 heavy chain KINH 109.60 0.99 6 1.00 0.29 29.07 inesin-like protein KIF11 KIF1 1 1 19.07 0.99 3 1.00 0.32 31 .72

Kinesin-Hke protein

KIF21A KI21A 187.04 1.00 6 1.00 0.46 45.67

Kinesin-like protein KIFC3 KIFC3 92.70 0.78 2 1.00 0.44 43.91

Krueppel-like factor 16 KLF16 25.40 0.98 2 1.00 0.35 35.15

Krueppel-related zinc

finger protein 1 HKR1 75.06 0.87 2 LOO 0.50 50,25

Latrophilin-2 LPHN2 163.23 0.97 4 1.00 0.44 43.95

Leu cine-rich repeat and IQ

domain-containing protein

1 LRIQ1 199.16 0.85 2 1.00 0.34 33.63

Leucine-rich repeat- containing protein 43 LRC43 72.96 0.84 2 1.00 0,46 45.60

Leucine-rich repeat- containing protein 48 LRC48 61.00 0.98 3 1.00 0.34 33.54

LINE- 1 type transposase

domain-containing protein

1 LITD1 98.77 0.99 3 LOO 0.39 39.06

LON peptidase N-terminal

domain and RING finger

protein 1 LONF1 86.65 0.96 2 1.00 0.43 43.43

Lysos omal -as s oc i ated

transmembrane protein 4B LAP4B 41.10 0.73 2 1.00 0.38 38.14

Macrophage erythroblast

attacher MAEA 45.24 0.92 2 LOO 0.33 32.55

Mdm2-binding protein MTBP 102.1 1 0.87 2 LOO 0.50 49.68

Membrane-associated

guanylate kinase, WW and

PDZ domain-containing

protein 3 MAGI3 165.49 0.94 2 LOO 0.18 17.84

Membrane-associated

phosphatidylinositol

transfer protein 2 PITM2 148.82 0.81 2 1.00 0.34 33.63

Membrane-associated

progesterone receptor

component 1 PGRC1 21.64 0.88 2 LOO 0.78 77.72

Methyltransferase-like

protein 6 METL6 33.21 1.00 2 LOO 0.38 37.83

Microtubule-actin cross - linking factor 1 , isoforms

1/2/3/5 MACF1 837.77 1.00 7 LOO 0.40 40.13

Microtubule-associated

serine/threonine-protein

kinase 2 MAST2 196.30 0.89 2 LOO 0.53 52.58

Misshapen-like kinase 1 MINK I 149.71 0.98 3 LOO 0.28 28.06

Mitogen-activated protein

kinase kinase kinase 1 M3K1 164.35 1.00 3 LOO 0.56 56.07 mRNA cap guanine-N7

methyltransferase MCES 54.79 0.90 3 LOO 0.83 82.98 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa Probability Guidelines) Intensity stdev stdev mRNA-capping enzyme MCE1 68.49 0.88 2 1.00 0.44 44.18

Mucin- 19 MUC19 596.66 0.99 4 1.00 0.28 27.94

Mucin-2 MUC2 539.94 0.95 2 1.00 0.29 28.82

Multidrug resistance- associated protein 4 MRP4 149.41 0.92 2 1.00 0.19 19,43

Multiple epidermal growth

factor-like domains protein

8 MEGF8 302.88 0.99 2 1.00 0.17 16.53

Myb-related transcription

factor, partner of profilin MYPOP 42.46 0.87 3 1.00 0.43 42.67

Myosin phosphatase Rho- interacting protein MPRIP 1 16.44 0.95 3 1.00 0.27 26.63

Myosin-1 MYH1 222.99 0.74 2 1.00 0.44 43.83

Myotubularin-related MTMR

protein 8 8 78.85 0.74 2 1 .00 0.28 27.61

Na(+) H(+) exchange

regulatory cofactor NHE- RF 1 NHRF1 38.83 1.00 5 1.00 0.26 26.24

N-alpha-acetyltransferase

15, NatA auxiliary subunit NAA15 101.19 0.98 3 1.00 0.49 49.24

Nebulette NEBL 116.36 0.93 3 1.00 0.73 72.56

Nephrocystin-l NPHP1 83.23 0.90 2 1.00 0.59 59.22

Neurogenic locus notch

homolog protein 2 NOTC2 265.21 0.98 4 1.00 0.14 13.62

Neuroligin-3 NLGN3 93.82 0.87 2 1.00 0.42 41.62

NFAT activation molecule

1 NFAMl 29.65 0.94 2 1.00 0.43 43.1 1

Niemann-Pick CI protein NPC1 142.06 0.74 2 1.00 0.29 28.62

Nuclear pore complex

protein Nup205 NU205 227.76 0.89 3 1.00 0.37 37.02

Nuclear receptor ROR- alpha RORA 62.98 0.98 2 1.00 0.46 45.79

Nucleolar complex protein

2 homolog NOC2L 84.85 0.77 2 1.00 0.45 44.78

Nucleolar protein 14 NOP 14 97.59 0.95 3 1.00 0.50 50.18

Oligoribonuclease,

mitochondria! ORN 26.80 0.84 2 1.00 0.75 75.16

O-phosphoseryl-tRNA(Sec)

selenium transferase SPCS 55.67 0.83 3 1 .00 0.11 1 1 .31

Otoancorin OTOAN 128.43 0.96 2 1.00 0.38 38.28

Otoferlin OTOF 226.59 0.96 3 1.00 0.90 90.43

Ovostatin homolog 1 OVOS1 134.40 1.00 3 1.00 0.64 63.70

Oxidation resistance protein

1 OXR1 97.89 0.98 3 1.00 0.29 28.51

P protein P 92.77 0.89 2 1.00 0.52 51.74

PDZ and LIM domain

protein 5 PDLI5 63.89 0.99 2 1.00 0.52 52.47 Total

Protein Total Peptides

Weight Group (Paris % rel

Seauence Name Gene ID (kDa.) Probability Guidelines) Intensity stdev stdev

Peroxisomal proliferator- activated receptor A- interacting complex 285

kDa protein PR285 294,45 0.84 3 1.00 0.13 12.70

Phospholipase D l PLD1 124.09 1.00 4 1.00 0.38 38.37

Piwi-like protein 1 PIWL1 98.52 1.00 2 1 ,00 0.32 3 1 .59

Plastin-1 PLSI 70.19 0.76 2 1.00 1 .07 106.63

Pleckstrin homology

domain-containing family

A member 3 PK iA.3 33.82 0.96 2 1 ,00 0.45 44.82

Pleckstrin homology

domain-containing family

H member 1 PKHH1 1 51.12 0.92 3 1.00 0.81 80.52

Poly [ADP-ribose]

polymerase 6 PARP6 71.05 0.78 2 1.00 0.24 24.44

Polycystic kidney disease

and receptor for egg jelly- related protein PKDRE 255.27 0.94 2 1.00 0.45 45.14

Polymeric immunoglobulin

receptor PIGR 83.21 0.98 3 1.00 0.64 63.86

POTE ankyrin domain

family member A POTEA 56.1 1 0.91 3 1.00 0.32 3 1 .77

PRAME family member 2 PRAM2 54.82 0.87 2 1.00 0.58 58.37

Probable ATP-dependent

RNA helicase DDX27 DDX27 89.76 1.00 5 1.00 0.41 41.41

Probable ATP-dependent

RNA helicase YTHDC2 YTDC2 160.13 0.85 3 1 .00 0.30 29.65

Probable G-protem coupled

receptor 1 10 GP 1 10 101.28 0.84 2 1 .00 0.35 34.64

Probable G-protein coupled

receptor 179 GP 179 257.18 1.00 5 1.00 0.41 40.86

Probable histone-lysine N- methy!transferase NSD2 NSD2 152.14 1.00 4 1.00 0.24 24.45

Probable phospholipid- transporting ATPase IC AT8B 1 143.59 0.90 2 1.00 0.58 58.20

Probable phospholipid- transporting ATPase II A ATP9A 1 18.49 0.91 2 1 .00 0.36 36.01

Probable ribonuclease

ZC3H12B ZC12B 92.97 0.94 2 1 .00 0.15 1 5.19

Prostamide/prostaglandin F

synthase PGFS 21.19 1.00 2 1 .00 0.43 43.35

Protein AATF AATF 63.08 0.90 3 1 .00 0.71 71.07

Protein FAM169B F 169B 21.38 0.72 2 1.00 0.52 52.37

Protein FAM81 A FA81A 42.35 0.97 2 1 .00 0.50 49.58

Protein FAM83H FA83H 127.03 0.97 3 1.00 0.21 20.96

Protein phosphatase 1

regulatory subunit 12B MYPT2 1 10.32 0.88 2 1.00 0.15 15.27

Protein phosphatase 1

regulatory subunit 16A ΡΡΪ 6Α 57.76 0.95 2 1 .00 0.28 28.47 Total

Protein Totai Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa) Probability Guidelines) Intensity stdev stdev

Protein phosphatase 1

regulatory subunit 32 PPR32 47.25 0.79 2 1.00 0.16 15.67

Protein piccolo PCLO 552.92 1.00 5 1.00 0.26 25.61

Protein PRRC2A PRC2A 228.71 0.96 3 1.00 0.13 12.99

Protein ripply3 DSCR6 20.34 0.83 2 1.00 0.37 37.35

Protein Shroom2 SHRM2 176.28 0.93 2 1.00 0.43 42.83

Protein SREK1 IP1 SRI IP 18.15 0.95 2 1.00 0.16 16.05

Protein TANC2 TANC2 219.49 0.93 2 1.00 0.35 35.45

Protein transport protein

Sec24C SC24C 1 18.23 0.89 2 1.00 0.37 36.79

Proto-oncogene DBL MCF2 107.59 0.99 3 1.00 0.50 50.00

Putative pre-mRNA- splicing factor ATP- dependent RNA helicase

DHX16 DHX16 1 19.17 0.90 2 1.00 0.32 31.63

Putative RNA polymerase

II subunit Bl CTD

phosphatase RPAP2 RPAP2 69.45 0.91 3 1.00 0.71 70.60

Putative zinc finger protein

705G Z705G 34.72 1.00 2 1.00 0.42 41.63

Pyruvate dehydrogenase

phosphatase regulatory

subunit, mitochondrial PDPR 99.28 0.96 2 1.00 0.46 46.14

R3H and coiled-coil

domain-containing protein

1 R3HC1 53.21 0.97 2 1.00 0.18 17.66

Ras GTPase-activating

protein nGAP NGAP 128.46 0.71 2 1.00 0.19 18.70

Ras GTPase-activatmg-iike

protein IQGAP1 IQGA1 189.12 0.90 2 1.00 0.64 64.30

RAS protein activator like-

3 RASL3 1 1 1.81 0.99 3 1.00 0.21 21.13

Ras-related protein Rap- lb RAPI B 20.79 0.99 2 1.00 0.39 38.91

Receptor expression- enhancing protein 3 REEP3 29.23 0.95 2 1.00 0.52 52.26

Receptor-interacting

serine/threonine-protein

kinase 1 RIP I 75.86 0.90 2 1.00 0.41 40.60

Receptor-type tyrosine- protein phosphatase C PTPRC 147.14 0.79 2 1.00 0.55 54.92

Regulator of nonsense

transcripts 3 A REN3A 54.65 0.96 3 1.00 0.62 61.76

Replication protein A 70

kDa DNA-binding subunit RFAl 68.08 0.85 3 1.00 0.41 41.48

REST corepressor 1 RCOR1 52.98 0.94 3 1.00 0.38 38.38

Reticulon-3 RTN3 1 12.52 0.75 2 1.00 0.22 21.78

Retinoic acid-induced

protein 1 RAI1 203.21 1.00 3 1.00 0.15 15.17

Rho GTPase-activating

protein 28 RHG28 81.99 0.82 3 1.00 0,25 25.35 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID fkDa) Probability Guidelines) Intensity stdev stdev

Rho guanine nucleotide

exchange factor 19 ARHGJ 89.12 0.76 2 1.00 0.39 39.45

Ribosome maturation

protein SBDS SBDS 28.73 0.98 3 1.00 0.29 29.20

RING finger protein

unkempt-like UNKL 73.76 0.89 2 1.00 0.42 41 ,57

RNA methyltransferase- like protein 1 RMTL1 46.97 0.86 3 1.00 0.42 42.41

RNA-binding protein 39 RBM39 59.32 0.79 2 3 .00 0.19 19.49

RNA-binding protein 43 RBM43 40.62 0.92 2 1.00 0.44 44.23

RNA-binding protein Raly RALY 32.43 0.86 2 1.00 0.45 45.07

Rotatin RTTN 248.45 0.94 3 1.00 0.10 10.24

Runt-related transcription

factor 2 RUNX2 56.59 0.93 3 1.00 0.27 27.48

S I RNA-binding domain- containing protein 1 SRBD1 1 1 1.69 0.99 4 1.00 0.29 29.46

Sarcop 1 asm ic/endoplas m ic

reticulum calcium ATPase

1 AT2A1 1 10.16 0.99 3 1.00 0.66 66.32

Selenium binding protein 1, Q9H8A

isoform CRA b 8 25.92 0.99 2 1.00 0.95 94.97

Semaphorin-3F SEM3F 88.31 0.99 2 1 .00 0.71 70.79

Serine/arginine-rich

splicing factor 6 SRSF6 39.55 0.81 3 1.00 0.60 59.99

Serine/threonine-protein

kinase 2C ST32C 54.94 1.00 2 1.00 0.70 70.36

Serine/threonine-protein

kinase tousled-like 2 TL 2 87.59 0.75 2 1.00 0.39 39.41

Serine/threonine-protein

kinase WNK1 WNK1 250.62 0.89 2 1.00 0.13 13.13

Serine/threonine-protein

kinase WNK2 WNK2 242.51 0.91 2 1.00 0.17 16.75

Serine/threonine-protein

phosphatase 4 regulatory

subunit 4 PP4R4 99.37 0.97 3 1.00 0.48 47.95

Serpin A12 SPA 12 47.13 0.79 2 1 .00 0.30 30.19

Signal-induced

proliferation-associated 1- like protein 3 SI1L3 194.47 0.94 2 1.00 0.31 1.46

Ski oncogene SKI 79.94 0.98 2 1.00 0.56 56.23

Sodium-dependent

phosphate transporter 1 S20A1 73.63 0.84 2 1.00 0.40 40.29

Solute carrier family 2

(Facilitated glucose Q504W

transporter), member 11 2 54.38 0.94 2 1.00 0.35 34.54

Solute carrier family 22

member 6 S22A6 61.76 0.99 2 1.00 0.18 1 7.58

Sortilin-related receptor SORL 248.25 0.99 2 1 .00 0.32 31.82

Sorting nexin-14 SNXI4 1 10.09 1.00 2 1.00 0.32 32.06 Total

Protein Total Peptides

Weight Group (Paris % rel

Seauence Name Gene ID fkDa Probability Guidelines Intensity stdev stdev

Spectrin alpha chain,

erythrocyte SPTA1 279.82 0.95 4 1.00 0.3 1 31.30

Spermatid-specific linker

histone Hl -like protein HILS1 25.60 0.90 2 1 .00 0.30 30.50

Sperm-specific antigen 2 SSFA2 138.28 0.72 2 1.00 0.89 89.01

Sphingomyelin

phosphodiesterase 4 NSMA3 93.27 0.99 3 1.00 0.34 34.28

Splicing factor 1 SF01 68.27 0.86 2 1.00 0.32 3 1.68

Structvfrai maintenance of

chromosomes protein 5 SMC5 128.71 0.97 2 1.00 0.50 50.28

Structural maintenance of

chromosomes protein 6 SMC6 126.23 0.94 3 1.00 0.34 33.91

Synemin SYNEM 172.65 0.75 2 1 .00 0.23 22. 1

Syntaxin-1 1 STX 1 1 33.16 0.93 2 1 .00 0.23 23.26

Talin-2 TLN2 271.43 1.00 4 1 .00 0.25 24.80

Tankyrase-1 TNKS 1 141 .93 0.98 3 1.00 0.44 43.96

Target of rapamycin

complex 2 subunit

MAPKAPl SIN1 59.07 0.71 2 1.00 0.36 35.92

TBC1 domain family

member 15 TBC 15 79.42 0.82 2 1.00 0.40 40.12

T-box transcription factor

TBX3 79.32 0.99 2 1.00 0.42 41 .55

Tektin-5 56.24 0.92 2 1.00 0.27 27.23

Telomerase protein

component 1 TEPl 290.29 0.83 2 1.00 0.73 73.36

Tetratricopeptide repeat

protein 21 B TT21B 150.82 0.99 3 1 .00 0.14 13.80

Tetratricopeptide repeat

protein 35 TTC35 34.79 0.87 2 1 .00 0.28 28.28

Tetratricopeptide repeat

protein 40 TTC40 303.29 0.99 5 1.00 0.28 28.23

TNFAIP3 - interacting

protein 2 TNIP2 48.65 0.92 2 1.00 0.44 43.79

Transcription termination

factor 2 TTF2 129.49 0.95 2 1.00 0.43 43.04

Transcriptional protein

SWT1 SWT1 103.14 0.91 2 3 .00 0.44 43.88

Transient receptor potential

cation channel subfamily M

member 5 TRPM5 131.35 1.00 4 1.00 0.27 26.77

Trans lational activator

GCN1 GCN1L 292.55 0.91 3 1.00 0.43 42.62

Transmembrane prolyl 4- hydroxylase P4HTM 56.61 0.73 2 1 .00 0.24 23.66

Transmembrane protein

131 -like T131 L 179.21 0.80 2 1.00 0.29 29.1 1

Transmembrane protein

132C T132C 121.69 0.95 3 1 .00 0.57 57. 14 Total

Protein Total Peptides

Weieht Group (Paris % rel

Sequence Name Gene ID fkDa) Probability Guidelines) Intensity stdev stdev

Transmembrane protein

C9orfl25 CI125 46.54 0.90 2 1.00 0.35 35.24

Q96KH

Transporter 8 70.48 0.97 2 1.00 0.43 42.86 tRNA (adenine(58)-N(l ))- methyitransferase non- catalytic subunit TRM6 TRM6 55.75 0.96 4 1.00 0.25 24.52 tRNA (guanine(37)-Nl)- methyltransferase TRM5 58.19 0.92 2 1.00 0.67 67.41 tRNA (guanine-N(7)-)- methyltransferase subunit

WDR4 WDR4 45.44 0.92 2 1.00 0.45 44.83 tRNA guanos ine-2'-0- methy transferase TRMl 3

homolog TRM13 54.19 0.91 2 3 .00 0.53 52.76

Tudor domain-containing

protein 7 TDRD7 123.49 0.80 2 1 .00 0.58 58.18

Q2M3B

Tyrosine hydroxylase 55.59 0.91 2 1.00 0.47 46.57

Tyrosine-protein

phosphatase non-receptor

type 13 PTN13 276.71 0.96 3 1.00 0.62 62.42

U2 small nuclear

ribonucleoprotein auxiliary

factor 35 kDa subunit- related protein 1 U2AFL 57.59 0.99 3 1.00 0.28 27.51

Ubiquitin carboxyl-terminai

hydrolase 19 UBP19 145.54 0.85 2 1.00 0.18 17.81

Ubiquitin carboxyl-terminai

hydrolase 31 UBP31 146.54 1.00 4 1.00 0.21 20.82

UDP-N-acetylhexosamine

pyrophosphorylase UAP1 58.71 0,96 2 1.00 0.25 24.89

Unconventional myosin-If MYOIF 124.75 0.99 4 1.00 0.55 55.47

Vacuolar protein sorting- associated protein 35 VPS35 91.63 0.77 2 1.00 0.35 35.20

WASH complex subunit

strumpellin STRUM 134.18 0.99 2 1.00 0.26 26.26

WD repeat-containing

protein 60 WDR60 122.48 0.99 4 1.00 0.53 52.65

WD repeat-containing

protein 87 WDR87 332.96 0.99 4 1.00 0.60 59.72

Xaa-Pro aminopeptidase 1 XPP1 69.86 0.91 2 1.00 0.53 53.41

Zinc finger BED domain- containing protein 1 ZBED 78.09 0.99 4 1.00 0.42 42.47

Zinc finger CCCH domain- containing protein 18 ZCH18 106.30 0.99 5 1.00 0.20 20.41

Zinc finger homeobox

protein 2 ZFHX2 273.99 0.92 4 1.00 0.37 36.85

Zinc finger protein 1 14 ZN1 14 47.70 0.71 2 1.00 0.29 29.19

Zinc finger protein 1 3 ZN133 73.32 0.98 4 1.00 0.55 55.06 Total

Protein Total Peotides

Weight Groao (Paris % rel

Sequence Name Gene ID OiDa) Probability Guidelines) Intensity stdev stdev

Zinc finger protein 143 ZN143 68.84 0.80 2 1.00 0.79 78.94

Zinc finger protein 441 ZN441 80.06 0.85 2 1.00 0.75 74.82

Zinc finger protein 484 ZN484 98.14 0.85 2 1.00 0.31 30,57

Zinc finger protein 512B Z512B 97.18 0.98 4 1.00 0.32 31.64

Zinc finger protein 57

homolog ZFP57 51.87 0.85 3 1.00 0.66 66.13

Zinc finger protein 619 ZN619 63.26 0.93 2 1.00 0.55 55,37

Zinc finger protein 638 ZN638 220.47 0.91 2 1.00 0.06 5.55

Zinc finger protein 671 ZN671 60.92 0.97 2 1.00 0.86 86.27

Zinc finger protein 717 ZN717 105.17 0.93 2 LOO 0.23 22.73

Zinc finger protein 778 ZN778 81.89 0.99 3 1.00 0.41 40.56

Zinc phosphodiesterase

ELAC protein 2 RNZ2 92.14 0.82 2 1.00 0.32 32,35

Zygote arrest protein 1 ZAR1 45.83 0.98 2 1.00 0.37 36.98

[Pyruvate dehydrogenase

[Iipoamide]] kinase

isozyme 3, mitochondrial PDK3 46.89 0.91 2 1.00 0.46 45.56

A disintegrin and

metal ioproteinase with

thrombospondin motifs 19 ATS 19 133.94 0.99 4 1.00 0.20 19.89

ADP-ribosylation factor- like protein 13B AR13B 48.59 0.98 2 1.00 0.32 32.13

Calpain-15 CAN 15 1 17.22 1.00 2 1.00 0.49 48.85

Chromosome 1 open

reading frame 49 Q5T0J8 24.50 0.76 2 1.00 0.69 69.09

Cleavage and

polyadenylation specificity

factor subunit 3 CPSF3 77.42 0.95 2 1.00 0.32 31.95

Dedicator of cytokinesis

protein 2 DOCK2 21 1.79 0.99 3 1.00 0.15 15.13

DnaJ homolog subfamily A

member 2 DNJA2 45.70 0.84 2 1.00 0.22 22.49

E3 ubiquitin-protein ligase

SMURF2 SMUF2 86.12 0.89 2 1.00 0.23 22.98

Glutamate receptor,

ionotropic kainate 5 GRI 5 109.18 0.99 4 1 .00 0.86 86, 10

Glutathione S-transferase

C-terminal domain- containing protein GSTCD 71.01 1.00 3 1.00 0.43 43.45

Growth/differentiation

factor 5 GDF5 55.36 0.88 2 1.00 0.81 81.24

Hepatocyte growth factorlike protein HGFL 80.25 0.74 2 1.00 0.27 27.35

IQ domain-containing

protein C IQCC 52.98 0.80 2 1.00 0.46 45.59

LIM domain-binding

protein 1 LDB1 46.48 0.92 2 1.00 0.48 48.07

Lysine-rich coiled-coil

protein 1 KR.CC I 30.94 0.80 2 1.00 0.30 29.89

Metailothionein- 1 G MT1G 6.12 0.89 2 1.00 0.40 40.06 Total

Protein Total Peptides

Weight Grouo (Paris % rel

Sequence Name Gene ID Probability Guidelines) Intensity stdev stdev

Metastasis-associated

protein MTA3 MTA3 67.44 0.91 3 1.00 0.46 45.99

Muscarinic acetylcholine

receptor M2 ACM2 51 .66 0.98 3 1.00 0.15 15.49

Nicotinate-nucieotide

pyrophosp orylase

[carboxyiating] NADC 30.81 0.92 2 1.00 0.47 47.17

Nidogen-1 NIDI 136.27 0.95 2 1 .00 0,38 37.70

Period circadian protein

homolog 3 PER3 131.79 1.00 2 1.00 0.27 27.02

Phosphoinositide 3-kinase

regulatory subunit 5 PI3R5 97.27 0.81 2 1.00 0.53 52.90

Poly(A)-specific

ribonuclease PARN PARN 73.39 0.91 2 1.00 0.16 16.40

Polypeptide N- acetylgalactosaminyltransfe

rase 2 GALT2 64.67 0.95 3 1.00 0.60 60.30

Probable helicase with zinc

finger domain HELZ 218.81 0,78 2 1.00 0.26 26.49

Proenkephalin-B PDYN 28.35 0.99 3 LOO 0.66 65.79

Protein KIBRA KIBRA 125.21 0.91 2 LOO 0.49 48.82

Protein NOV homolog NOV 39.12 0.90 2 LOO 0.1 8 18.38

Reeiin RELN 388.12 0.92 3 LOO 0.31 30.51

Serpin B 13 SPB I 3 44.23 0.89 2 LOO 0.33 32.84

TBCI domain family

member 2A TBD2A 105.33 0.99 2 LOO 0.40 40.23

Trafficking protein particle

complex subunit 12 TPC12 79.31 0.72 2 LOO 0.55 54.81

UPF0614 protein

C14orfl 02 CN102 132.57 0.94 3 1.00 0.22 22.06

V-type proton ATPase

subunit B, kidney isoform VATB1 56.78 LOO 4 1 .00 0.35 34.92

WD repeat-containing

protein 52 WDR52 1 1 1.64 0.98 2 1.00 0.25 25.33

YLP motif-containing

protein 1 YLPM1 219.83 0.99 ₂ 1.00 0.45 44.68

Zinc finger CCCH domain- containing protein 13 ZC3HD 196.50 1.00 6 1.00 0.39 39.23

Zinc finger protein 30 ZNF30 71.35 0.82 2 1.00 0.28 27.55

Polycystic kidney disease

protein l-like 2 PK1L2 272.38 0.96 4 1.00 0.35 34.78

SCO-spondin SSPO 547.12 0.90 3 LOO 0.43 42.67

UPF0490 protein Cl orGO I CA201 36.74 1.00 4 1.00 0.17 16.78

26S protease regulatory

subunit 6A PRS6A 49.15 1.00 3 LOO 0.18 18.22

A disintegrin and

metalloproteinase with

thrombospondin motifs 4 ATS4 90.12 0.89 2 1 .00 0.37 36.83 Totai

Protein Total Peptides

Weieht Group (Paris % rel

Secjuence Name Gene ID fkDa Probability Guidelines Intensity stdev stdev

A disintegrin and

metal loproteinase with

thrombospondin motifs 5 ATS5 101.63 0.94 2 1.00 0.36 35.79

Achaete-scute associated Q8WZ5

protein 8 39.05 0.98 4 1.00 0.22 21.68

A-kinase anchor protein 2 AKAP2 94.58 0.87 3 1.00 0.83 83.24

Alanine— tRNA ligase,

cytoplasmic SYAC 106.73 0.94 3 1.00 0.31 31.14

Alpha- 1,6- mannosylglycoprotein 6- beta-N- acety!glucosaminyitransfera

se B MGT5B 89.46 0.75 2 1.00 0.37 36.85

Annexin E5RK69 51.73 0.98 3 1.00 0.40 40.44

APC membrane recruitment

protein 1 AMER1 123.93 1.00 6 1.00 0.19 19.37

Armadillo repeat- containing protein 3 ARMC3 96.33 0.87 2 1.00 0.52 51.96

Aspartyl/asparaginyl beta- hydroxylase ASPH 85.79 0.79 2 1.00 0.19 18.67

A TP-binding cassette subfamily C member 9 ABCC9 174.09 0.77 2 1.00 0.49 49.55

ATP-binding cassette subfamily D member 1 ABCDl 82.87 0.99 2 1.00 0.50 50.52

Axin-1 AXIN1 95.56 0.83 2 1.00 0.09 8.56

BMP-2-inducible protein

kinase BMP2K 129.07 0.94 3 1.00 0.49 49.46

B7ZM1

C3orf30 protein 5 60.01 0.98 3 1.00 0.37 36.60

Calbindin B7Z9J4 23.58 1.00 2 1.00 0.49 49.18

Calcium/calmodit! independent protein kinase

type 11 subunit gamma CC2G 62.55 0.95 2 1.00 0.49 49.46

Calcium-activated chloride

channel regulator 4 CLCA4 101.20 0.98 2 1.00 0.30 29.75

Cellular retinoic acid- binding protein 1 RABP1 15.54 0.71 2 1.00 0.47 46.98

Centrosomal protein of 135

kDa CP135 133.39 1.00 2 1.00 0.16 16.49

Cirhin CIR1A 76.82 0.95 2 1.00 0.27 26.59

Collagen alpha-l (XVII)

chain COHA1 150.31 0.99 2 1.00 0.30 30.46

Cyclophilin-33B Q3S61 1 34.98 0.73 2 1.00 0.20 19.92

Cytochrome c oxidase

subunit 4 isoforra 1 ,

mitochondrial COX41 19.55 0.78 2 1 .00 0.30 30.33

D -beta-hydroxybutyrate

dehydrogenase,

mitochondrial BDH 38.1 1 0.85 3 1.00 0.43 43.45 Total

Protein Totai Peptides

Weight Group (Paris % rel

Seauence Name Gene ID (kDa) Probability Guidelines) Intensity stdev stdev

Dedicator of cytokinesis

protein 9 DOCK9 236,28 1.00 2 1.00 0.42 41.81

Dynein heavy chain 6,

axonemal DYH6 475.66 0.97 5 1.00 0.45 44.88

E3 ubiquitin-protein ligase

NEDD4 NEDD4 149.00 0.96 3 1.00 0.28 27.55

Endothelial zinc finger

protein induced by tumor

necrosis factor alpha ZNF71 54.44 1.00 2 1.00 0.21 20.88

Fanconi anemia group D2

protein FACD2 366.34 0.99 2 1.00 0.19 19.51

Forkhead box protein SI FOXS1 35.39 0.84 2 1.00 0.18 18.06

Formin-1 FMN1 157.46 0.99 3 1.00 0.09 9.47

G patch domain-containing

protein 8 GPTC8 164.08 0.98 3 1 .00 0.84 84.09

Glutaminase liver isoform,

mitochondrial GLSL 66.26 0.98 3 1.00 0.38 37.88

Histone-lysine N- methyltransferase PRDM9 PRDM9 103.29 0.96 2 LOO 0.37 36.64

Histone-lysine N- methyitransferase, H3

lysine-36 and H4 lysine-20

specific NSD1 296.45 0.99 3 1.00 0.56 55.75

Hypermethylated in cancer

2 protein HIC2 66.10 1.00 4 1.00 0.52 52.12

Immunoglobultn-like

domain-containing receptor

2 ILDR2 71.14 0.83 2 1.00 0.18 17.64

Inactive phospholipase C- like protein 1 PLCL1 122.63 0.79 2 1.00 0.37 36.61

Interleukin- 1 receptor- associated kinase 1 IRAKI 76.47 0.82 2 1.00 0.66 66.12

B7ZLX

ITGAV protein 0 1 12.15 1.00 2 1.00 0.31 31.48

KRR1 small subunit

process ome component

homo log KRR1 43.62 0.98 2 1.00 0,38 38.54

Lamin-B l LMNB1 66.35 0.73 2 1.00 0.29 28.69

Laminin subunit alpha-2 LAMA2 343.67 0.97 4 1.00 0.44 43.62

Letha!(3)malignant brain

tumor-like protein 4 LMBL4 71.06 0.94 2 1.00 0.39 39.35

Leucine zipper putative

tumor suppressor 2 LZTS2 72.70 0.97 2 1.00 0.83 83.55

Leucine-rich repeat- containing protein 10 LRC10 31 .60 0.86 2 1.00 0.50 50.21

LIXl-like protein LIX1L 36.52 0.75 2 1.00 0.84 84.01

Low-density lipoprotein

receptor-related protein 2 LRP2 521.60 1.00 4 LOO 0.12 1 1.64

MAP7 domain-containing

protein 2 MA7D2 81.90 0.79 2 LOO 1.19 1 19.02 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa> Probability Guidelines') Intensity stdev stdev

Mediator of DNA damage

checkpoint protein 1 MDCi 226.51 0.96 3 1.00 0.07 6.95

Membrane protein

F AM 174 A F174A 19.92 0.83 2 1.00 0.48 48, 14

B9EGR

MGA protein 5 3 4.92 1.00 6 1.00 0.87 87.09

Myosin- 8 MYH8 222.61 0.93 2 1.00 0.42 41.61

NACHT, LRR and PYD

domains-containing protein

4 NALP4 1 13.32 0.98 3 1.00 0.44 44.23

Netrin receptor UNC5C UNC5C 103,06 0.98 3 1.00 0.54 54.15

Neugrin NGRN 32.37 0.94 2 1.00 0.35 35.21

Neurobeach in-like protein 2 NBEL2 302.31 0.73 2 1.00 0.81 80.77

Nuclear pore complex

protein Nup214 NU214 213.47 0.98 3 1.00 0.15 14.88

Outer dense fiber protein 2 ODFP2 95.32 0.98 2 1.00 0.72 72.42

Pericentrin PCNT 377.79 0.99 5 1.00 0.33 32.75

Peroxisome biogenesis

factor 1 PEX1 142.76 0.95 3 1 .00 0.53 52.96

PEST proteolytic signal- containing nuclear protein PCNP 18.90 0.79 2 1.00 0.58 57.98

Pleckstrin homology

domain-containing family

N member 1 PKHN1 71.73 0.98 3 1.00 0.60 59.77

Pleiotrophin PTN 18.91 0.78 2 1.00 0.44 43.87

Pre-mRNA-process ing

factor 40 homo log A PR40A 108.72 0.90 2 1.00 0.32 32.04

Probable ATP-dependent

RNA helicase DDX46 DDX46 117.27 0.85 3 1.00 0.45 44.75

Protein dopey-2 DOP2 258.05 0.79 3 1.00 0.44 43.98

Protein FAM131 B F131B 35.73 0.94 2 1.00 0.41 40.57

Protein RRP5 homolog RRP5 208.55 1.00 4 1.00 0.34 33.67

Protein unc-79 homolog UNC79 295.10 0.97 3 1.00 0.26 26.18

Protein Wnt-1 1 WNT1 1 39.14 0.99 3 1.00 0.46 46.53

Protein-glutamine gamma- glutamyltransferase 6 TGM3L 79.24 0.78 2 1.00 0.37 36.97

Proteoglycan 4 PRG4 150.97 0.88 3 3 .00 0.61 61.27

Protocadherin Fat 3 FAT3 505.19 0.83 2 1.00 0.53 53.02

Pumilio domain-containing

protein C14orf21 CN021 69.38 0.99 2 1.00 0.80 79.75

Putative PRAME family

member 25 54.26 0.92 2 1.00 0.32 32.52

Pyruvate kinase isozymes

M1 M2 PYM 57.88 0.89 2 1.00 0.38 38.22

RELT-like protein 2 RELL2 32.37 0.99 3 1.00 0.26 25.74

Retinal dehydrogenase 1 AL1A1 54.81 0.94 2 1.00 0.38 37.97

RNA po!ymerase- associated protein CTR9

homolog CTR9 133.40 0.74 2 1 .00 0.53 52.90

RNA -bind ing protein 10 RBMI O 103.45 0.99 4 1.00 0.30 29.60 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa) Probability Guidelines) Intensity stdev stdev

RRP12-like protein RRP12 143.59 0.98 3 1.00 0.36 35.82

Sarcoplasmic/endoplasmic

reticulum calcium ATPase

3 1 13.89 0.95 3 LOO 0.74 74.07

Q1RMY

SEMA6C protein 5 59.30 0.92 3 1.00 0.64 63.93

Semaphorin-3C SEM3C 85.13 0.78 2 1.00 0.31 31 ,40

Semaphorin-5A SEM5A 120.52 0.87 2 1.00 0.35 35.22

Serine/arginine-rich

splicing factor 1 1 SRS 1 1 53.49 0.99 3 1.00 0.27 27.38

Serine/ arginine-r ich

splicing factor 4 SRSF4 56.63 1.00 4 1.00 0.69 69.26

S erine/threonine-protein

kinase pim-1 PIM1 45.37 0.94 2 1.00 0.45 44.77

Serine/threonine-protein

kinase PLK2 PLK2 78.17 0.88 2 1.00 0.52 51.90

Serine/threonine-protein

kinase PL 4 PL 4 108.89 0.90 3 1.00 0.28 27.80

Serine/threonine-protein

kinase TAOl TAO 1 1 15.98 0.97 3 1.00 0.68 68.21

Small G protein signaling

modulator 3 SGSM3 85.28 0.92 3 1.00 0.55 55.28

SNARE-associated protein

Snapin SNAPN 14.85 0.83 3 1.00 0.29 29.23 snRNA-activating protein

complex subunit 3 SNPC3 46.70 0.95 2 1.00 0.46 46.00

Sodium channel protein

type 1 1 subunit alpha SCNBA 204.77 0.77 2 1.00 0.57 57.1 1

Spermatogenesis-associated

serine-rich protein 2 SPAS2 59.49 0.88 3 1.00 0.97 97.00

Spondin-1 SPON1 90.90 0.87 3 1.00 0.30 29.94

Sulfotransferase 1C3 ST1C3 35.85 0.90 2 1.00 0.79 78.95

Symplekin SYMPK 141.04 0.93 2 1.00 0.26 25.70

Syncoilin SYNCI 55.25 0.99 3 1.00 0.65 65.12

Tetratricopeptide repeat

protein 28 TTC28 270.70 0.88 2 1.00 0.20 19.91

Tetratricopeptide repeat

protein 31 TTC31 57.05 0.87 2 1.00 0.24 23.60

T-!ymphoma invasion and

metastasis -inducing protein

1 TIAMl 177.38 0.99 4 1.00 0.73 72.94

TRAF-interacting protein

with FHA domain- containing protein A TIFA 21.41 0.86 2 1.00 0.46 46.42

Transcription elongation

factor B polypeptide 3 ELOA1 89.84 0.88 3 1.00 0.59 58.70

Transcription regulator

protein BACH2 BACH2 92.46 0.87 2 1.00 0.33 32.62

Transmembrane protein

184A T184A 45.73 0.94 2 1.00 0.38 38.21 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa) Probability Guidelines) Intensity stdev stdev

Transmembrane protein 62 TMM62 73.07 0.92 2 1.00 0.33 32.61

Treslin TICRR 210.71 0.94 4 1.00 0.44 44.33

Q8N4C

TRPV5 protein 1 43.12 0.99 3 1.00 0.76 76.03

Ubiquitin-associated

protein 1 UBAP1 55.03 0.95 2 1.00 0.58 57.76

Unconventional myosin-Ic MYO C 121.59 0.97 2 1.00 0.10 10.28

Unconventional myosin- XVIIIb MY18B 284.99 0.79 2 1.00 0.30 29.95

UPF0583 protein CI 5orf59 CO059 31.89 0.82 2 1.00 0.27 27.14

Vascular endothelial

growth factor receptor 3 VGFR3 152.64 1.00 4 1.00 0.30 30.39

WD repeat-containing

protein 19 WDR19 151.47 0.83 2 1.00 0.26 26.16

YTH domain family protein

3 YTHD3 63.80 0.93 2 1.00 0.44 44.30

Zinc finger CCCH-type

with G patch domain- containing protein ZGPAT 57.31 0.74 2 1.00 0.22 22.33

Zinc finger CCHC domain- containing protein 2 ZCHC2 125.84 0.97 2 1.00 0.20 19.81

Zinc finger FYVE domain- containing protein 16 ZFY16 168.78 0.85 2 1.00 0.42 41.77

Zinc finger protein 2 ZNF2 48.66 0.71 2 1.00 0.48 48.34

Zinc finger protein 205 ZN205 60.57 0.81 2 1.00 0.45 44.62

Zinc finger protein 292 ZN292 304.61 0.86 2 1.00 0.35 35.14

Zinc finger protein 330 ZN330 36.16 0.98 3 1.00 0.39 39.05

Zinc finger protein 473 ZN473 100.10 0.79 2 1.00 0.53 52.96

Zinc finger protein 512 ZN512 64.62 0.99 2 1.00 0.30 30.08

Zinc finger protein 608 ZN608 162.09 0.99 4 1.00 0.11 10.59

Zinc finger protein 684 ZN684 43.90 0.73 2 1.00 0.33 33.01

Zinc finger protein 718 ZN718 55.35 0.99 2 1.00 0.48 48.05

Zinc finger protein 750 ZN750 77.29 0.88 2 1.00 0.43 42.62

Zinc finger protein 816 ZN816 75.66 0.75 2 1.00 0.43 43.51

26S proteasome non- ATPase regulatory subunit

12 PSD12 52.85 0.94 3 1.00 0.53 52.88

80 kDa MCM3 -associated MCM3

protein A 218.25 1.00 2 1.00 0.78 78.45

ATPase family AAA

domain-containing protein

5 AT AD 5 207.42 0.97 3 1.00 0.43 43.12

ATP-binding cassette subfamily F member 2 ABCF2 71.23 0.98 2 LOO 0.39 39.36

BRCA1 -associated protein BRAP 67.24 0.93 3 1.00 0.55 55.18

Carbohydrate

sulfotransferase 3 CHST3 54.65 0.96 2 1.00 0.58 58.55

C-C motif chemokine 15 CCL15 12.22 0.90 2 1.00 0.58 57.69

Centromere-associated

protein E F8W957 275.73 0.98 4 1.00 0.17 17.25 Total

Protein Total Peptides

Weight Group (Paris % rel

Sequence Name Gene ID (kDa) Probability Guidelines) Intensity stdev stdev

Condensin complex subiinit

1 B4E0B7 152.12 0.94 4 1.00 0.90 90.02

EGF-!ike and EMI domain- containing protein 1 EGFEM 21.60 0.93 2 1.00 0.33 32.82

Fer-l -like protein 5 FR1L5 241.79 1.00 3 1.00 0.37 37.28

Glycerophosphocho!ine

phosphodiesterase

GPCPD1 GPCP1 75.97 0.80 2 1.00 0.37 37.08

Histone H2B type F-M H2BFM 28.23 0.91 2 1.00 0.55 54.97

Immunoglobulin

superfami!y member 10 IGS10 290.64 0.97 4 1.00 0.18 18.42 integrator complex sub unit

6 INT6 100.31 1.00 3 1.00 0.66 66.05

Keich domain-containing

protein 10 LD10 49.05 0.94 2 LOO 0.33 33.49

Krueppel-like factor 5 LF5 50.74 0.76 2 LOO 0.47 46.70

Methionine-tRNA ligase,

cytoplasmic SYMC 101.03 0.80 2 LOO 0.29 28.79

Mitochondrial import

receptor subunit TOM5

homolog TOMS 6.01 1.00 2 LOO 0.39 39.34

M-phase phosphoprotein 8 MPP8 97.10 0.76 2 LOO 0.78 77.98

Nuclear receptor

corepressor 1 NCOR1 270.03 0.96 5 LOO 0.47 47.49

Oral-facial-digital

syndrome 1 protein OFDI 1 16.58 0.83 2 LOO 0.48 48.46

Peregrin BRPF1 137.39 0.91 3 LOO 0.18 17.78

PHD finger protein 23 PHF23 43.77 0.71 2 1.00 0.78 77.72

Protein kinase C-binding

protein NELL1 NELL1 89.56 0.94 3 1.00 0.60 60.34

Protein SERAC1 SRAC1 74.08 0.86 3 LOO 0.54 53.87

RAS guanyl-releasing

protein 2 GRP2 69.19 0.89 2 LOO 0.54 53.63

Rho-associated protein

kinase 2 ROC 2 160.78 0.97 3 LOO 0.19 18.86

SEC14-Iike protein 2 S14L2 46.10 0.99 3 LOO 0.89 89.28

Serine palmitoyltransferase

3 SPTC3 61.99 0.95 2 LOO 0.54 53.79

S erine/threon ine-protein

kinase haspin HASP 88.42 0,94 2 LOO 0.17 17.13

SH3 domain and

tetratricopeptide repeat- containing protein 2 S3TC2 144.67 0.94 2 LOO 0.40 39.86

Spectrin beta chain, brain 2 SPTN2 271.14 0.97 5 LOO 0.10 10.01

StAR-reiated lipid transfer

protein 9 STAR9 516.01 1.00 6 LOO 0.23 23.14 tRNA pseudouridine(38/39)

synthase PUS3 55.59 0.88 2 LOO 0.52 52.03

Vacuolar protein sorting- associated protein 37B VP37B 31.27 1.00 2 LOO 0.45 45.48 Total

Protein Total Peptides

Weight Group (Paris % rel

Seauence Name Gene ID fkDa t Probability Guidelines) Intensity stdev stdev

Zinc finger FYVE domain- containing protein 1 ZFYVI 87.10 0.90 2 1.00 0.40 40.00

Zinc finger protein 20 ZNF20 61.51 0.85 3 1.00 0.60 60.58

Zinc finger protein 334 Z 334 79.58 0.87 3 1.00 0.14 14.49

FYVE, RhoGEF and PH

domain-containing protein

6 FGD6 160.70 0.79 2 1.00 0.62 62.28

Leucine zipper protein 1 LUZP1 120.18 1.00 4 1 .00 0.40 40.37

Multiple epidermal growth

factor-like domains protein

9 MEGF9 62.92 0.86 2 1.00 0.13 12.77

5-methyltetrahydrofolate- homocysteine B7ZLW

met yltransferase 8 140.49 1.00 3 1.00 0.58 58.13

Zinc finger protein 101 ZN101 50.29 0.92 3 1.00 0.61 61.40

Zinc finger protein 385B Z385B 50.36 0.95 2 1.00 0.33 33.52

Zinc finger protein 385D Z385D 0.92 3 1.00 0.33 33.52

Coiled-coil domain- containing protein 12 CCD 12 19.15 0.97 3 1.00 0.35 35.24

5268