BIOMARKERS FOR MYOCARDIAL ISCHEMIA

This application claims the benefit of the filing date of provisional patent application 61/128,688, filed May 23, 2008, which is incorporated by reference in its entirety herein.

BACKGROUND INFORMATION

Coronary heart disease is the most common single cause of death in the western world, representing about 20% of all deaths. This is equivalent to about 2 million deaths in Europe per year or four people per minute. In the US, over 8 million people exhibit acute (chest pain) symptoms in the Emergency Department (ED) of hospitals, with 1.5 million individuals having confirmed acute coronary symptom (ACS) events, accounting for 500,000 short term deaths. In patients presenting to the emergency room with chest pain, fewer than 15% are ultimately diagnosed as having ischemia or acute myocardial infarction (MI). Currently, blood tests for the cardiac specific isoform of troponin I or troponin T (TnI or TnT, respectively) are generally used for the diagnosis of acute myocardial infarction (due to cardiac muscle (cell) death). Creatine kinase (CK) MB and myoglobin can also be used, but are considered to be less specific for cardiac injury. However, although these cardiac biomarkers can identify patients with even small amounts of myocardial necrosis, there is an earlier time point in which the heart is in ischemia but is not yet in necrosis, and the diagnosis of cardiac ischemia in the absence of necrosis cannot currently be made with accuracy.

It would be useful to be able to identify subjects in this diagnostic window (having non- necrotic ischemia). Such a diagnostic tool would be of great value for triage in the emergency department. For example, it would allow earlier intervention, including earlier perfusion, to allow increased salvage of the injured myocardium; and it would prevent unnecessary admittance to the hospital of patients with non-cardiac chest pain. Furthermore, such an assay could delay therapy in subjects who do not exhibit diagnostic electrocardiographic (ECG) changes, and could help to improve the accuracy of current provocative tests for ischemia, such as exercise stress testing. The sooner intervention can be carried out, the less cardiac damage will occur. Reduced damage is correlated with an increase in long term survival.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic representation of protein spot attrition due to stringent criteria, based on a cohort that underwent atrial pacing to induce demand on the heart such that the blood

flow was inadequate, thereby potentially caus ing myocardial ischemia. Blood samples were drawn from the coronary sinuses of individuals subjected to atrial pacing. The change in lactate acid level was an indication of induced ischemia, while the detection of cTnl or cTnT was indicative of cardiac necrosis. Those subjects that did not exhibit any change in lacate acid level or cTnT/cTnT were considered controls.

Figure 2 shows a 2DE gel image showing two spots that are elevated in necrosis individuals. Serum samples were initially depleted of immunoglobins (IgG) and albumin, then separated based on pi and MW using gel electrophorisis. The majority of the spots did not change in all of the individuals subjected to atrial pacing. The graphs on the right show the spot volume in all three necrosis individuals. For both spots, 2 out of 3 were increased for those indivudials which had cTnl detected after atrial pacing in their coronary sinus blood samples. Figure 3 shows IDLC fractions strategy used in MS analysis. Samples were depleted of immunoglobins (IgG, IgM and IgA) and albumin, then separated based on hydrophobicity (Reversed phase high performance liquid chromatography, RPLC, IDLC). The number of unique peptides/protein observed, and the number of times observed and protein coverage are semi-quantitative, because each domain will comprise the same protein species, although potentially differing in amounts.

Figure 4 shows the number of peptides observed vs. the number of spectra count for all individuals and all time points obtained by IDLC (hydrophobicity , RPLC - MS run 1). Figure 5 schematically illustrates the overlap between the three different proteomic methods used: 2DE, IDLC and 2DLC. For 2DLC, the serum samples were depleted of immunoglobins (IgG, IgM and IgA) and albumin, then separated based on chromatographic focusing (pH from 8.5 to 4.0) and reversed phase HPLC (I DLC). Fractions from areas found to be different in the 2DLC were analyzed by MS. Differences in spectral counting and or number of peptides observed were included as changed. Superscripts 1 -6 refer to the following:

1. Two additional proteins were observed in each protein spot. Caspase 14 was observed to change in AMI (individual) 15 and by IDLC for individuals (1-10) that underwent atrial pacing

2. Most stringent - greater than 2 fold increase (3.2) in 2/3 ischemia or AMI patients as well as fluctuation in other individuals 3. Reduced stringency (fold change reduced (2.2)) greater than 1.5 fold change

4. Reduced stringency - greater than 2 fold increase in 2/3 ischemia or AMI patients but fluctuates with individuals

5. LPLNECA-I = isoform 1 of Long palate, lung and nasal epithelium carcinoma- associated protein 1 6. LMW and HMW kinnogen-1 were detected but it was the bradykinin peptide that was elevated

Figure 6 shows schematically the collection during valve replacement. In this chorot, coronary sinus samples were obtained from individuals who underwent induced ischemia due to stopping of the heart (with cardioplegia) during valve replacement. Coronary sinus samples were obtained and depleted prior to being separated by IDLC (as outlined above). Proteins found to be increased with ischemia in the majority of indivuals were considered first teir. However, it must be recognized that lower abundant proteins may only be observed in a few patients due Io inherent detection limits of this type of MS analysis. These proteins might be actually elevated in many patients and just not observed with this approach.

Figure 7 shows a box plot of cTnl at all time points for the individuals that under went valve replacement. Note that all indivudals eventually had detectable cTnl/cTnT in their serum, indicating necrosis. However, at the time points at which de novo discovery was undertaken, none of the individuals had detectable cTnT or cTnl. This shows that all were ischemic at the time of study. DESCRIPTION

The present inventors have identified a number of protein markers for cardiac (myocardial) ischemia, including non-necrotic cardiac (myocardial) ischemia.

Three different types of protein analysis were performed to identify these markers, in order to cover as broad a base as possible of proteome coverage, e.g. to allow the enhanced detection of isoforms and of post-translational modifications (PTM). These types of analysis were two-dimensional electrophoresis (2DE, separating proteins based on pi and molecular weight), two-dimensional liquid chromatography (2DLC, separating proteins based on pi and hydrophobicity) and one-dimensional liquid chromatography (IDLC, separating proteins based on hydrophobicity). Note that the starting pH differs between 2DLC and 1 DLC: pH 8.5 and 2.3, respectively. Two different cohorts were analyzed - increased metabolic demand (cohort 1) and reduced supply (cohort 2).

In a first study, ischemia was induced in a first cohort of subjects by metabolic demand: subjects were stimulated by atrial pacing, which makes the heart beat faster and induces ischemia, as indicated by an increase in lactate, and potentially myocardial necrosis (based on detection of cTnl or cTnT in blood). In some cases, individuals did not exhibit any increase in lactate or detectable cTnl/cTnT. These latter individuals were considered controls. Multiple serum samples were obtained for each individual. Differences between baseline (prior to pacing) and those at peaking pacing and up to 60 minutes after were analyzed. Those proteins that were

elevated compared to the baseline in the majority of ischemic or necroiss indivuals (and not elevated in controls) were considered to be of interest.

This procedure mimics naturally occurring metabolic cardiac events, such as ministrokes, that might precede a full MI. Ischemia is a heterogeneous group of conditions, resulting from different underlying mechanisms, such as demand and supply limitation. We have "mimicked" these two conditions in the different cohorts used in the analysis. Thus, these cohorts are expected to reflect markers that are overexpressed in subjects suffering from ischemia resulting from a variety of such underlying mechanisms. Samples from demand (atrial pacing) were evaluated by 2DE, 2DLC and IDLC. In a second study, ischemia was induced in a second cohort by coronary blockage: subjects undergoing valve replacement surgery exhibited ischemia because of blood loss during the procedure. This procedure mimics naturally occurring events in which ischemia is induced by coronary blood vessel blockage. This cohort was evaluated only by IDLC, the procedure which provided a comparison to the most useful results with the first cohort. Those proteins found to be altered in both cohorts are considered to be "tier one" markers, although strong hits in either cohort may also be considered to be prime candidate markers.

The results of the studies with these two cohorts arc summarized in Table 13. Taken together, these studies show that three proteins are implicated as the most highly correlated markers (sometimes referred to herein as "first tier" markers, as they are observed to be elevated in both cohorts) for ischemia, regardless of the cause of the ischemia: Lumican; Extracellular matrix protein 1 (ECM-I); and Carboxypeptidase N (e.g., the catalytic chain).

Three markers in addition to Lumican, ECM-I and Carboxypeptidase N are implicated as first tier markers for at least subjects similar to those in the first cohort: Angiogenin; Semenogclin (e.g., isoforms 1 and 2); and Long palate, lung and nasal epithelium carcinoma- associated protein I (LPLNECA-I) (e.g., isofonn 1 ; isoforms 2-4 are also present, but the method of analysis employed in this study, although it supports isoform 1, cannot distinguish among the four isoforms, which are splice variants, so isoforms 2-4 cannot be ruled out due to sequence homology).

Ten markers in addition to Lumican, ECM-I and Carboxypeptidase N are implicated as first tier markers for at least subjects similar to those in the second cohort: Perioxiredoxin isoform 2; SlOO isoforms A7, λ8 and A9 (other S lOO isoforms were detected and not observed to be altered); Sortilin-related receptor; Catalase; Low density lipoprotein receptor related proteins 1 and 2; and Syntaxin 3.

In addition to these first tier markers, Table 13 lists some "second tier" markers which can also be used for identifying subjects similar to those in both cohorts I and II; in cohort I; or in cohort II. These include, e.g., Alpha-2-HS-glycoprotein; Galectin-7; Hornerin; Proteoglycan- 4; Profϊlaggrin (also called Filaggrin); Vitamin D binding protein; C4b-binding protein alpha chain; Thyroxine binding globulin; Alpha-2-glycoprotein 1, zinc; protease, serine 3; Caspase 14; Desmogelin; Kininogen-1 (we observed the peptide for the intact protein, but our data cannot distinguish between changes to the LMW or HMW, which could also be present); Hepatocyte growth factor like protein; Hepatocyte growth factor activator; and Insulin like growth factor protein 6. In some embodiments of the invention, it is desirable to distinguish between subjects whose ischemia is induced by metabolic causes (similar to the subjects of cohort I), and subjects whose ischemia is induced by coronary blood vessel blockage (similar to the subjects of cohort II), because different treatment methods can be used for the two classes of subjects. The markers of the invention can be used to make such distinctions.

This invention relates, e.g., to a method for determining if a subject has myocardial ischemia, comprising measuring in a sample from the subject the amount of at least one of the following proteins, compared to a baseline value: a) Lumican and/or b) Extracellular matrix protein 1 and/or c) Carboxypeptidase N, wherein a significant amount (e.g., at least a statistically significant amount) of over- expression of the protein(s) compared to the baseline value is indicative of myocardial ischemia (e.g., indicates that the subject has, or is likely to have, myocardial ischemia). The amount of expression may be determined for any combination of 1 , 2, or all 3, of these proteins, and the determinations can be conducted simultaneously, or in any order.

Another aspect of the invention is a method for identifying subjects that have myocardial ischemia that is induced by a metabolic-induced ischemic event [due to a metabolic limitation, in which the heart is unable to meet metabolic need; (excessive) metabolic demand], comprising determining in the sample from the subject the amount, compared to a baseline value, of at least one of proteins a), b), c) above, d) Angiogenin, e) Semenogclin, and/or

f) Long palate, lung and nasal epithelium carcinoma-associated protein 1. The amount of expression may be determined for any combination of 1, 2, 3, 4, 5, or 6 of these proteins, and the determinations can be conducted simultaneously, or in any order.

Another aspect of the invention is a method for identifying subjects that have myocardial ischemia that is induced by coronary blood vessel blockage, which limits the supply of blood, comprising determining in the sample from the subject the amount, compared to a baseline value, of at least one of proteins a), b), c) above, g) Syntaxin, h) Perioxiredoxin isoform 2, i) S100 isoform A7,

J) SlOO isoform A8, k) SlOO isoform A9,

1) Sortilin-rclated receptor m) Catalase n) Low density lipoprotein receptor related protein 1, and/or o) Low density lipoprotein receptor related protein 2. The amount of expression may be determined for any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 of these proteins, and the determinations can be conducted simultaneously, or in any order.

In addition to the proteins noted above, one of more of the "second tier" proteins indicated in Table 13 can also be measured. A skilled worker will recognize which of lhese markers are indicative of a cohort I-typc of condition, and which are indicative of a cohort II- type of condition.

Another aspect of the invention is a method for determining if a subject has myocardial ischemia, comprising determining in a sample from the subject the amount, compared to a baseline value, of at least one (e.g., at least four) of at least proteins a) - p) as noted above. The amount of expression may be determined for any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of these proteins; and the determinations can be conducted simultaneously, or in any order.

In a method of the invention, a determination that increasing numbers of protein markers of the invention are overexpressed in a subject can further indicate that the subject has (or is likely to have) myocardial ischemia.

A method as above may further comprise measuring in the sample the amount of one or more other markers that have been reported to be diagnostic of cardiac necrosis, including

cardiac specific isoforms of troponin I (TnI) and/or troponin T (TnT) (although CK-MB, myoglobin, have been used in the past, cTnl and cTnT are the current gold standards), wherein a significant increase (e.g., at least a statistically significant increase) of the one or more markers further is further indicative that the subject has myocardial ischemia. As noted above, ischemia is a heterogeneous condition caused by a variety of underlying mechanisms. Even if a single marker of the invention is capable of detecting a subject having ischemia resulting from a particular mechanism, it is possible for some markers that the marker is also upregulated in a disease other than myocardial ischemia. In such a case, it would be desirable to screen for upregulation of at least one additional marker that is associated with ischemia caused by a different underlying mechanism. The column labeled "Function" in Table 13 shows that some of the markers of the invention can be divided into particular groups on the basis of their functions. A skilled worker, studying this table, could readily identify markers associated with different mechanisms. In one embodiment of the invention, markers associated with 2, 3, 4 or more underlying mechanisms can be tested together in an assay of the invention. Another aspect of the method is a method for deciding how to treat a subject suspected of having myocardial ischemia, or a subject that is at high risk for having myocardial ischemia, comprising determining by a method as above if the subject has (or is likely to have) myocardial ischemia and, (1) if the subject is determined to have (or to be likely to have) myocardial ischemia, deciding to treat the subject aggressively [such as with angioplasty (mechanical widening in opening blood vessels), treating with an anti-thrombolysis agent or, if possible, with percutaneous coronary intervention (PCI, or TPA), or undergoing coronary bypass surgery to replace the injuried/blocked coronary artery], or (2) if the subject is determined not to have (or not to be likely to have) myocardial ischemia, deciding to treat the subject non-aggressively [such as with asprin and/or thrombolysis (e.g., TPA), with periodic monitoring Io ensure no future MI events, or by recommending changes in life style. This method can be used to confirm that a subject dpes not have ischemia (especially if myocardial ischemia is not detectable by cTnl or cTnT elevation), and thus to allow the subject to be released from hospital care]

Another aspect of the invention is a method for treating a subject suspected of having myocardial ischemia, or a subject that is at high risk for having myocardial ischemia, comprising determining by a method as above if the subject has (or is likely to have) myocardial ischemia and, (1) if the subject is determined to have (or to be likely to have) myocardial ischemia, treating the subject aggressively, as indicated above, or (2) if the subject is determined not to

have (or not to be likely to have) myocardial ischemia, treating the subject non-aggressively, as indicated above.

Another aspect of the invention is a kit for detecting the presence of ischemia in a subject, comprising reagents for detecting the amounts of at least one (e.g., any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 of at least proteins a) - o) as noted above.

This invention relates, e.g., to a method for determining if a subject has myocardial ischemia, comprising

(a) providing a sample obtained from a subject suspected of having myocardial ischemia; (b) determining in the sample the amount of at least one of at least proteins a) - p) as noted above (e.g., any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of the proteins); and

(c) comparing, the amount(s) of the protein(s) to a baseline value that is indicative of the amount of the protein in a subject that does not have myocardial ischemia, wherein an increased amount (e.g., a statistically significantly increased amount) of the protein(s) compared to the baseline value is indicative of myocardial ischemia.

In one embodiment of the invention, the amount(s) of the protein(s) is compared over time to the baseline value and/or to levels known to be associated with necrosis. The kinetic rise and fall of combinations of proteins is indicative of impending myocardial ischemia (or other cardio and vascular events, such as stroke. A method of the invention can also be used to determine risk in subjects (patients) with stable or unstable angina.

A sample which is "provided" can be obtained by the person (or machine) conducting the assay, or it can have been obtained by another, and transferred to the person (or machine) carrying out the assay.. By a "sample" (e.g. a test sample) from a subject meant a sample that might be expected to contain elevated levels of the protein markers of the invention in a subject having myocardial ischemia. Many suitable sample types will be evident to a skilled worker. In one embodiment of the invention, the sample is a blood sample, such as whole blood, plasma, or serum (plasma from which clotting factors have been removed). For example, peripheral, arterial or venous plasma or serum can be used. In another embodiment, the sample is urine, sweat, or another body fluid into which proteins are sometimes removed from the blood stream. In the case of urine, for example, the protein is likely to be broken down, so diagnostic fragments of the proteins of the invention can be screened for. In another embodiment, the sample is cardiac

tissue, which is harvested, e.g., after a heart transplant or the insertion of a pacemaker or defibrillator. Methods for obtaining samples and preparing them for analysis (e.g., for detection of the amount of protein) are conventional and well-known in the art. Some suitable methods are described in the Examples herein or in the references cited therein. A "subject," as used herein, includes any animal that has, or is suspected of having, myocaridal ischemia. Suitable subjects (patients) include laboratory animals (such as mouse, rat, rabbit, guinea pig or pig), farm animals, sporting animals (e.g. dogs or horses) and domestic animals or pets (such as a horse, dog or cat). Non-human primates and human patients are included. For example, human subjects who present with chest pain or other symptoms of cardiac distress, including, e.g. shortness of breath, nausea, vomiting, sweating, weakness, fatigue, or palpitations, can be evaluated by a method of the invention. About 1/4 of MI are silent and without chest pain. Furthermore, patients who have been evaluated in an emergency room or in an ambulance or physician's office and then dismissed as not being ill according to current tests for infarction have an increased risk of having a heart attack in the next 24-48 hours; such patients can be monitored by a method of the invention to determine if and when they begin express markers of the invention, which indicates that, e.g., they are beginning to exhibit ischemia. Subjects can also be monitored by a method of the invention to improve the accuracy of current provocative tests for ischemia, such as exercise stress testing. An individual can be monitored by a method of the invention during exercise stress tests of Dobutamine stress tests to determine if the individual is at risk for ischemia; such monitoring can supplement or replace the test that is currently carried out. Athletes (e.g., humans, racing dogs or race horses) can be monitored during training to ascertain if they are exerting themselves too vigorously and are in danger of undergoing an MI.

In another embodiment of the invention, the method is used as a screen in order to identify a drug (or to improve a cardioplegic solution) that protects the heart from ischemia and necrosis. The detection of one or more of the proteins of the invention in blood (or media if cell culture is used) is indicative of ischemia, and the quantity of the protein(s) is indicative of the severity of the ischemia.

The properties and amino acid sequences of the proteins of the invention are well-known and can be determined routinely, as well as downloaded from various known databases. See. e.g., the database, International Protein Index (IPI) at the world wide web site, ebi.ac.uk/IPI/xrefs.html. A summary of some properties of some of the proteins discussed herein, including their IPI ID number and amino acid sequences, is provided in Examples II and

IV. This information is accurate as of the date of filing of this application. However, some of this information, including the sequences, is routinely updated (e.g. to correct mistakes in the previous entries), so updated (corrected) information about the proteins is included in this application. Information provided in the FPI database is incorporated by reference in the present application.

Although much of the data presented in the Examples herein are directed to particular forms of proteins of interest (or peptides thereof), it will be evident to a skilled worker that a variety of forms of these proteins may be indicative of the presence of myocardial ischemia in a subject. For example, the protein may be an intact, full-length protein. If a protein undergoes processing naturally (e.g., is converted from a pre-pro-hormone to a pro-hormone to a fully processed hormone; the N-terminal methionine is cleaved off; the signal sequence is removed, often accompanied by a post-translational modification, such as acetylation; etc.), any of these forms of the protein are included in the invention. Furthermore, in some instances, a protein of the invention may be broken down or degraded (e.g., proteins that are found in the urine). In such a case, an investigator can determine the level of one or more of the fragments or degradation products. A "diagnostic protein fragment," as used herein, is a fragment that is unique to the protein being identified, as detected by the assay. For example, a diagnostic fragment is recognized specifically by an antibody used to detect the full-length protein. Certain isoforms or post translational modifications (PTM) may also be encompassed by the invention. For example, the inventors have obtained data indicating PTM for C4b binding proteins; protease, serine; 3 alpha-2-glycoprotein 1; and zinc caspase 14.

The proteins and combinations of proteins discussed herein are sometimes referred to herein as "proteins (or protein markers) of the invention."

A variety of tests that have been used to detect myocardial events (particularly late occurring events, such as necrotic myocardial ischemia). These include, e.g., determining the levels of cardiac specific isoform(s) of troponin I (TnI) and/or troponin T (TnT), CK-MB (Creatine Kinase-MB), or myoglobin, although only the former two are the current gold standard. CK MB and myoglobin are not cardiac-specific. However, none of these markers is completely satisfactory for the detection of myocardial ischemia _. . For example, they fail to detect early stages of heart disease, such as non-necrotic myocardial ischemia. The new markers described herein can be used in conjunction with these types of assays.

When the values of more than one protein are being analyzed, a statistical method such as multi-variant analysis or principal component analysis (PCA) is used which takes into

account the levels of the various proteins (e.g., using a linear regression score). For verification, we will use either immunoassay or multiple reaction monitoring (MRM, a MS-based targeted method that quantifies peptides that are unique to the protein of interest) on individuals (control, ischemia and MI). In some embodiments, it is desirable to express the results of an assay in terms of an increase (e.g., a statistically significant increase) in a value (or combination of values) compared to a baseline value.

A "significant" increase in a value, as used herein, can refer to a difference which is reproducible or statistically significant, as determined using statistical methods that are appropriate and welϊ-known in the art, generally with a probability value of less than five percent chance of the change being due to random variation. In general, a statistically significant value is at least two standard deviations from the value in a "normal" healthy control subject. Suitable statistical tests will be evident to a skilled worker. For example, a significant increase in the amount of a protein compared to a baseline value can be about 50%, 2-fold, or more higher. A significantly elevated amount of a protein of the invention compared to a suitable baseline value, then, is indicative that a test subject has myocardial ischemia (indicates that the subject is likely to have myocardial ischemia). A subject is "likely" to have myocardial ischemia if the subject has levels of the marker protein(s) significantly above those of a healthy control or his own baseline (taken at an earlier time point). The extent of the increased levels correlates to the % chance. For example, the subject can have greater than about a 50% chance, e.g., greater than about 70%, 80% 90%, 95% or higher chance, of having the ischemia. In general, the presence of an elevated amount of a marker of the invention is a strong indication that the subject has ischemia.

As used herein, a "baseline value" generally refers to the level (amount) of a protein in a comparable sample (e.g., from the same type of tissue as the tested tissue, such as blood or serum), from a "normal" healthy subject that does not exhibit myocardial ischemia. If desired, a pool or population of the same tissues from normal subjects can be used, and the baseline value can be an average or mean of the measurements. Suitable baseline values can be determined by those of skill in the art without undue experimentation. Suitable baseline values may be available in a database compiled from the values and/or may be determined based on published data or on retrospective studies of patients' tissues, and other information as would be apparent to a person of ordinary skill implementing a method of the invention. Suitable baseline values may be selected using statistical tools that provide an appropriate confidence interval so that

measured levels that fall outside the standard value can be accepted as being aberrant from a diagnostic perspective, and predictive of ischemia.

It is generally not practical in a clinical or research setting to use patient samples as sources for baseline controls. Therefore, one can use any of variety of reference values in which the same or a similar level of expression is found as in a subject that does not have myocardial ischemia.

It will be appreciated by those of skill in the art that a baseline or normal level need not be established for each assay as the assay is performed but rather, baseline or normal levels can be established by referring to a form of stored information regarding a previously determined baseline levels for a given protein or panel of proteins, such as a baseline level established by any of the above-described methods. Such a form of stored information can include, for example, a reference chart, listing or electronic file of population or individual data regarding "normal levels" (negative control) or positive controls; a medical chart for the patient recording data from previous evaluations; a receiver-operator characteristic (ROC) curve; or any other source of data regarding baseline levels that is useful for the patient to be diagnosed. In one embodiment of the invention, the amount of the proteins in a combination of proteins, compared to a baseline value, is expressed as a linear regression score, as described, e.g., in Irwin, in Neter, Kutner, Nachtsteim, Wasserman (1996) Applied Linear Statistical Models, 4 ^th edition, page 295. In an embodiment in which the progress of a treatment is being monitored, a baseline value can be based on earlier measurements taken from the same subject, before the treatment was administered.

The amount of a protein can be measured using any suitable method. Some methods involve the use of antibodies, binding ligands, or mass spectrometry tagged peptides specific for a protein of interest. Antibodies suitable for use in assays of the invention are commercially available, or can be prepared routinely. Methods for preparing and using antibodies in assays for proteins of interest are conventional, and are described, e.g., in Green et al, Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), (Humana Press 1992); Coligan et al, in Current Protocols in Immunology, Sec. 2.4.1 (1992); Kohler & Milstein (1975), Nature 256, 495; Coligan et al, sections 2.5.1-2.6.7; and Harlow et al, Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Laboratory Pub. 1988).

Any of a variety of antibodies can be used in methods of the invention. Such antibodies include, e.g., polyclonal, monoclonal (mAbs), recombinant, humanized or partially humanized,

single chain, Fab, and fragments thereof. The antibodies can be of any isotype, e.g., IgM, various IgG isotypes such as IgGf IgGi ₈ , etc., and they can be from any animal species that produces antibodies, including goat, rabbit, mouse, chicken or the like. The term, an antibody "specific for" a protein, means that the antibody recognizes a defined sequence of amino acids, or epitope in the protein. An antibody that is "specific for" a polypeptide refers to an antibody that binds selectively to the polypeptide and not generally to other polypeptides unintended for binding to the antibody. The parameters required to achieve such specificity can be determined routinely, using conventional methods in the art. Conditions that are effective for binding a protein to an antibody which is specific for it are well-known and conventional. In one embodiment of the invention, antibodies specific for a (one or more) protein of the invention arc immobilized on a surface (e.g., are reactive elements on an array, such as a microarray, or are on another surface, such as used for surface plasmon resonance (SPR)-bascd technology, such as Biacore), and proteins in the sample are detected by virtue of their ability to bind specifically to the antibodies. Alternatively, proteins in the sample can be immobilized on a surface, and detected by virtue of their ability to bind specifically to the antibodies. Methods of preparing the surfaces and performing the analyses, including conditions effective for specific binding, are conventional and well-known in the art.

Among the many types of suitable immunoassays are immunohistochemical staining, ELISA, Western blot (immunoblot), immunoprecipitation, radioimmuno assay (RIλ), fluorescence-activated cell sorting (FACS), etc. Assays used in a method of the invention can be based on colorimetric readouts, fluorescent readouts, mass spectrometry, visual inspection, etc. Assays can be carried out, e.g., with suspension beads, or with arrays, in which antibodies or cell or blood samples are attached to a surface such as a glass slide or a chip.

In one embodiment, a tissue sample (e.g. a cardiac tissue sample) is stained with a suitable antibody in a conventional immunohistochemical assay for those proteins which are prcsnt in the myuocardium. Note that it can be difficult to obtain human tissue unless an individual is undergoing surgery or a routine biopsy (e.g. following heart transplantation), and such subjects are likely to be ischemic to some degree.

Mass spectrometry (MS) can also be used to determine the amount of a protein, using conventional methods. Some typical such methods are described in the Examples herein. Relative ratio between multiple samples can be determined using label free methods (as done in the present Examples), based on spectral count (and the number of unique peptides and the number of observation of each peptide). In the Examples herein, we used a LTQ-Orbitrap

LC/MS/MS instrument to obtain the data. Alternatively, quantitive data can be obtained using multiple reaction monitoring (MRM), most often carried out using a triple quadripole mass spectrometer. In this case, peptides that are unique to a given protein are selected in the MS instrument and quantified. Absolute quantification can be obtained if a known labeled synthetic peptide is used. For detailed methods see, e.g., Qin Fu and JE Van Eyk, in Clinical Proteomics: from diagnostics to therapy (Van Eyk JE and Dunn M, eds), Wiley and Son Press; Current Protocols in Molecular Biology, Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom-Up Proteomics Workflow, Gundry et al., chapter 10, 2009, in press)

In general, molecular biology methods referred to herein are well-known in the art and are described, e.g., in Sambrook el al., Molecular Cloning: A Laboratory Manual, current edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & sons, New York, NY.

A detection (diagnostic) method of the invention can be adapted for many uses. For example, it can be used to follow the progression of cardiac ischemia. In one embodiment of the invention, the detection is carried out both before (or at approximately the same time as), and after, the administration of a treatment, and the method is used to monitor the effectiveness of the treatment. A subject can be monitored in this way to determine the effectiveness for that subject of a particular drug regimen, or a drug or other treatment modality can be evaluated in a pre-clinical or clinical trial. If a treatment method is successful, the levels of the protein markers of the invention are expected to decrease.

A method of the invention can be used to suggest a suitable method of treatment for a subject. For example, if a subject is determined by a method of the invention to be likely to have myocardial ischemia, a decision can be made to treat the subject with an aggressive form of treatment; and, in one embodiment, the treatment is then administered. Suitable aggressive treatment modalities include, for example, angioplasty (mechanical widening to open blood vessels); treating with an anti-thrombolysis agent or, if possible, with percutaneous coronary intervention- (PCI, or TPA); or undergoing coronary bypass surgery to replace the injuried/blocked coronary artery. Methods for carrying out such treatments are conventional and well-known. By contrast, if a subject is determined not to be likely to have myocardial ischemia, a decision can be made to adopt a less aggressive treatment regimen; and, in one embodiment, the subject is then treated with this less aggressive forms of treatment. Suitable less aggressive forms of treatment include, for example, treatment with asprin and/or agents that bring about thrombolysis {e.g., TPA); periodic monitoring to ensure no future MI events; or recommending

changes in life style. A subject that does not have myocardial ischemia is thus spared the unpleasant side-effects associated with the unnecessary, more aggressive forms of treatment. By "treated" is meant that an effective amount of a drug or other anti-heart disease procedure is administered to the subject. An "effective" amount of an agent refers to an amount that elicits a detectable response (e.g. of a therapeutic response) in the subject.

One aspect of the invention is a kit for detecting whether a subject is likely to have myocardial ischemia, comprising one or more agents for detecting the amount of a protein of the invention. As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" protein of the invention, as used above, includes 2, 3, 4, 5 or more of the proteins. In addition, other markers for ischemia (e.g., as discussed elsewhere herein) can also be present in a kit. If mass spectrometry is to be used to measure protein levels, the following reagents can be included in the kit: known amounts of a labeled (e.g. stable istope) peptide (synthetic or recombinant) standard for each peptide to be assessed, separately or combined into a single mixture containing all peptides; optionally, a different peptide standard for assessing reproduciblty of the assay; and/or, optionally, dilutant and trypsin for preparation of the sample. If an antibody-based method is to be used to measure protein levels, the agents in the kit can encompass antibodies specific for the proteins. The kit may also include additional agents suitable for detecting, measuring and/or quantitating the amount of protein, including conventional analytes for creation of standard curves. Among other uses, kits of the invention can be used in experimental applications. A skilled worker will recognize components of kits suitable for carrying out a method of the invention.

Optionally, a kit of the invention may comprise instructions for performing the method. Optional elements of a kit of the invention include suitable buffers, containers, or packaging materials. The reagents of the kit may be in containers in which the reagents are stable, e.g., in lyophilized form or stabilized liquids. The reagents may also be in single use form, e.g., for the performance of an assay for a single subject. In one embodiment of the invention, the kit is a "home chest pain test kit," that can be used to test blood, urine, or other body fluids for the presence (and/or level) of protein markers of the invention. Thus, a patient who has been released from an Emergency Department (ED) or a cardiac ward, but who is at risk over the next about 48 hours, can take the test over time at home and, if the test produces positive results, return to the ED.

In the foregoing and in the following examples, all temperatures are set forth in uncorrected degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.

EXAMPLES

Example I. Identification of novel cardiac biomarkers that are rapidly released into the coronary sinus in response to cardiac ischemia, even in the absence of detectable myocyte necrosis A. Overview of the studies

Rapid atrial pacing has been reported to produce reversible and controlled myocardial ischemia, as measured by a coronary sinus lactate concentration that rises above arterial lactate concentration, in approximately 2/3 of patients with fixed epicardial coronary artery disease (>70% diameter stenosis in at least one coronary artery). (Dehmer et al. (1983) Am Heart J 106. 114-24; Markham et al. (1983) Am J Cardiol 51, 1589-94). Therefore, in the experiments shown in this Example, atrial pacing was used as the human demand ischemia model.

In the studies shown in this Example, three types of protein analysis were conducted to identify protein markers of the invention. There is normally less than 40% overlap (e.g., 3-5) in the proteins observed between the different types of analysis platforms. This is because every protein is intrinsically different with respect to its pi, hydrophobicity and mass. Furthermore, post translational modifications (PTM) alter the intrinsic nature of the protein and thus may display quite different separation or enrichment characteristics. As such, the choice of technology or group of technologies should be dictated by the characteristics of the proteins targeted by the experimental question. In the case of biomarker discovery (and based on the lessons learnt from the biomarker, cTnl), multiple protein separation strategies should (and do) increase proteome coverage.

The present inventors and collaborators have found that the combination of intact protein separation technologies of 2DE (two-dimensional electrophoresis), 2DLC (two-dimensional liquid chromatography) and IDLC (one-dimensional liquid chromatography) increases the proteome coverage while allowing enhanced detection of isofoππs and. PTM. 2DE of serum (and plasma) was optimized for separation by using pH 4-7 and 10% Bis-Tris gel (Graham et al. (20050) Proteomics 5, 2309-14; Fu et al. (2005) Proteomics 5, 2656-64), as were the liquid chromatography methods. The combination of chemical depletion and optimized 2DE conditions can achieve good reproducibility (~20% CV) (Fu et al. (supra)). Liquid chromatography (LC) separates proteins based on one or more of their intrinsic properties: mass

(size exclusion), isoelectric point (pi, chromatographic focusing or ion exchange), hydrophobicity (reversed phase) or affinity chromatography (bio-specificity). Our laboratory has optimized 2DLC combining chromatographic focusing and reversed phase HPLC with the commercial Beckman Coulter instrument, the Two-Dimensional Protein Fractionation (PF2D) system (McDonald et al. (2006) MoI Cell Proteomϊcs 5, 2392-411 ; Sheng et al. (2006) MoI Cell Proteomics 5, 26-34; Stasna et al. Protein separation: Liquid chromatography, In "Proteomic and Genomic Analysis of Cardiovascular Disease" (eds. Van Eyk JE and Dunn M) 2008 Wiley and Son Press, page 241). Briefly, samples are loaded onto the first dimension column (ion exchange column) at pH 8.5 in presence of urea and detergent and separated based on pi by decreasing the pH to 4.0 (Graham et al. (2006) {supra). Proteins that are bound tightly to the column or have a pi below 4.0 are cluted using 1 M salt. We found that including 20% isopropanol in the buffers can eliminate "artificial" binding of a subset of proteins to the first dimension. Fractions are collected throughout the chromatographic separation and each fraction is subsequently separated by reversed phase chromatography using a linear gradient composed of aqueous trifloroacetic acid (TFA), pH 2.3 and TFA/actronitrile, pH 2.3. The second dimension elution profile is monitored at 214 nm (peptide bonds) and is semi-quantitative. On average, fractions contain 1- 100 proteins in each peak (McDonald et al. (2006); Graham et al. (2006) (both supra)). These samples can be further analyzed by electrophoresis (IDE or 2DE) or analyzed directly by mass spectrometry (MS). If so, due to the complexity of the reversed phase fractions they must undergo further online LC separation prior to MS. An overview outlining the procerss is summarized in Fu et al. (2008) (supra).

B, Cohort information' for atria pacing human model - cohort I 1. Research Design Patients > 20 years old with stable exertional angina referred for cardiac catheterization were recruited Exclusion criteria were atrial fibrillation, valvular heart disease, prior coronary artery bypass surgery, depressed left ventricular systolic function, acute coronary syndrome, and/or left bundle branch block. As well, patients were excluded if they reported angina within 48 hours of the catheterization. 19 individuals were recruited. The study was approved by the Institutional Review Boards of UT Southwestern and Parkland Hospital. AU patients have signed written informed consent.

A 7 or 8 Fr Gorlin catheter was advanced to the coronary sinus from the right brachial vein. Coronary sinus, peripheral arterial, and peripheral venous serum samples were obtained

prior to start of the atrial pacing. The left atrium was paced at 20 beats/minute above the resting heart rate and this was increased every 3 minutes by 20 beats/minute until one of the following occurs: chest pain, AV block, or a heart rate of 160 beats/minutes is achieved. The patient was maximally paced at this rate for 3 minutes. At the end of the three-minute period, repeat blood samples were collected from the coronary sinus and peripheral artery. Repeat sampling from the coronary sinus was performed at 30 and 60 minutes after pacing termination.

Table 1. Timing line for serum sample collection

Location Baseline Immediate 30 minutes 60 minutes post- post- post- pacing pacing pacing

Coronary X X X X

Sinus Blood was immediately placed on ice and was transported to the processing center within

30 minutes of collection. Samples were centrifuged, serum (and plasma) separated, and specimens aliquoted into 100 μL tubes using an automated micropipette system. No samples were at room temperature for longer than 10 minutes. The longest duration between sample collection and freezing was less than one hour. Lactate and cardiac troponin T (TnT) was measured in heparinized plasma a (see table 2)

2. Cohort and experimental group designation

The cohort was designated based on the following criteria:

1 ) Cases (n= 19) Significant coronary artery disease (at least one vessel with a diameter stenosis > 70%) and coronary sinus lactate > arterial lactate after pacing (data not shown).

2) Controls are individuals with no or little change in lactate pre vs. post.

3) Moderate or severe ischemia: individuals with increase in lactate and are cTnT negative. > 4) Necrosis designation was for individuals with increase in lactate and are cTnT positive.

Table 2: coronary sinus values

TnT- TnT- TnT- TnT- Definition

PATIENT age csLpre csLpoβt csO cs1 cs2 cs 3

1 50 0.4 ^' 0.7 <0.01 <0.01 <0.01 <0.01 ml

2 40 0.8 0.6 <0.01 <0.01 <0.01 <0.01 C

60 0.7 0.7 <0.01 <0.01 <0.01 <0.01

Pre and post define samples taken at baseline and after maximum pacing

Definition defines, control (c) as no change in lactate and TnT negative, ischemia (I) as increase in lactate and TnT negative and differentiated in to moderate (ml) or severe (si); necrosis (n) TnT positive and excluded for LC analysis (but included for 2DE).

C. 2-dimensional gel electrophoresis analysis

1. 2DE cohort

All patients and all time points were analyzed.

2.2DE Methods

Serum was depleted of IgG using protein G affinity chromatography and depleted of albumin using our in-house affinity/chemical depletion method (Fu tt al. (2005) (supra). Protein concentration was determined using BCA assay (Pierce) for each depleted sample. 50 ug of each time point (baseline time point 1, 2 and 3) per individual was labeled with one of the three Cy dyes (Applied Biosy stems Inc.). As well, a pool sample was created from equal amounts of each time point of a single patient sample. For each individual, equal amount of two labeled sample (two time points) were mixed with the pool sample and then separated simultaneously using optimized pH 4-7 gel, followed by 10% Bis-Tris SDS PAGE. The gels were then imaged on a fluorescent gel imager at the Cy3, Cy5 and Cy2 wavelengths. Subsequently, the gels were stained with silver to allow visualization for spot picking. Gel images were analyzed by Ludesi lnc (.http://www.ludesi.com/). Gels were aligned, spots matched and quantified. For example, gel images were prepared for an individual that became ischemic or underwent necrosis with pacing. Comparisons were made between baseline and the other subsequent time points for each individual. To avoid a nondetected (zero) value 0.1 was added to all values.

3. Selection criteria.

Selection criteria for 2DE was based on analysis of all individual in each group (induced ischemia and induced necrosis) and are as follows: i) Equal or greater than 1.5 fold increase compared to time point 0 (baseline). ii) The spot volume was above (100 units) to allow protein identification by mass spectrometry. iii) The spot was resolved. iv) The change in the profile remains above baseline once elevated. v) A changed in 3 out of the 3 or 2 out of the 3 individuals in a designated group

(induced ischemia or induced necrosis) at any time point.

4. Results for 2DE

Approximately 1200 protein spots were resolved on each 2DE gel. Due to stringent criteria for cut offs, most protein spots were deemed not to change or biological variability was too great to be significant (Figure 1, see breakdown). Caspase 14 and complement factor B

(isoform 1) increased specifically in patients with necrosis while fibrinogen beta chain and desmoglein-1 increased in patients with severe ischemia and necrosis (table 3).

Table 3: • summary of changes detected by 2DE observed at large

Protein name baseline control ischemia moderate ischemia necrosis

Caspase-14 yes 1 out of 5 1 out of 3 0 out of 4 2 out of 3

Isoform 1 of

Complement factor B yes 1 out of 5 1 out of 3 0 out of 4 2 out of 3

Fibrinogen beta chain yes 2 out of 5 1 out of 3 2 out of 4 2 out of 3

Desmoglein-1 yes 2 out of 5 1 out of 3 2 out of 4 2 out of 3

The majority of proteins observed by 2DE, high abundant soluble proteins, do not change with induced ischemia or necrosis. Without wishing to be bound by any particular mechanism, Caspase 14 (IPI00013885) is proposed to be involved in the death receptor and granzyme B apoptotic pathways. It may act as a downstream signal transducer of cell death. Desmoglein-1 (1PI00025753) is a component of the cell desmosome junctions which are distinct plasma membrane domains. It has a single transmembrane domain. Desmosomes are the most

common type of intercellular junction in vertebrate epithelial cells but found in other cell types. This protein is part of a complex comprising plakophilin 1, plakophilin 2, desmoplakin, desmoglein 1, desmoglein 4, plakoglobin and comeodesmosin. Other proteins of the desmosome complex as well as caspase 14 are found by IDLC and 2DLC in a few individuals. Information sheets summarizing some of the properties of these and other proteins discussed herein are provided as Example II.

D. IDLC work flow 1. IDLC cohort Each individual time sample outlined below (table 4) was analyzed using reversed phase

HPLC. The control group samples were selected based on having similar prelacate concentrations compared to the Iwo disease groups.

2. IDLC Method

Serum was depleted using an affinity chromatography comprised of IgY antibodies specific for all forms of immunoglobins (IgG, IgA and IgM) and then depleted of albumin using our in-house affinity/chemical depletion method. This was done in order to reduce background of the chromatogram. Protein concentration was determined using BCA assay (Pierce) for each depleted sample. Samples were analyzed on the same IDLC columns. 50 ug of each sample was run (in duplicate) using our optimized gradient. One set was used for mass spectrometric analysis; the other set was stored at -8O ⁰ C. Fractions were collected into 96-well plates, stored at -8O ⁰ C until analyzed. Protein standard was run every morning to ensure good, consistent and reproducible performance of HPLC system. Extensive washing was carried out between runs to eliminate possible cross-over contamination. Chromatographic images were compared and regions/domains with acceptable intensities from each experimental sample were selected or combined (figure 5). Total 650 fractions (26 fractions per sample) were dried down, neutralized, and digested with trypsin. 50% of the digested sample was applied to the LC LTQ-Orbitrap MS.

For LC-MS/MS experiments on the LTQ-Orbitrap (ThermoFinnigan, San Jose, CA), peptides were dissolved in 6 μl resuspension buffer (4% acetonitrile in water with 0.1% formic acid). Samples (3 μl) were loaded onto a 75um x 10 cm BioBasic C18 column (New Objective, Wobυrn, MA). Peptides were eluted into an LTQ-Orbitrap (ThermoFinnigan, San Jose, CA) using an Agilent 1200 nano-LC system (Agilent, Santa Clara, CA). The HPLC gradient was 5% to 60% B (90% acetonitrile/watcr in 0.1% Formic acid) over 30 or 60 min depended on sample complexity. The mass spectrometer was operated in data-dependent mode in which every FT- MS scan (survey 350-2000 Da) was followed by MS/MS scans of the 5 most abundant ions.

All mass spectrometry data was analyzed according to a pipeline established and already used in our laboratory, designed to meet stringent criteria from the proteomics community. Data from the LTQ-Orbitrap was searched against the IPI databases where possible, using Sorcerer Sequest (Sagen, San Jose, CA). Search results were validated and analyzed using Scaffold (Proteome Systems, Portland, OR). Protein identifications based upon a single peptide observation were handled carefully through manual inspection of the tandem MS (MS/MS) spectra, BLAST searching of the sequence to ensure it matches only the reported protein, requiring a minimum of 8 amino acids and a peptide probability score of >0.9.

Data analysis was based on the following: peptide redundancy removed, protein name redundancy removed, the number of unique peptides and number of observation for each peptide regardless of charge state (2+. 3+ or 4+) were determined for each protein. The protein was proposed to have a potential PTM if it was identified in multiple non-sequential domain/fractions. This was noted and all data regardless of the fraction was included for quantitation of the protein.

Data reanalysis was carried out using a version of the published algorithm for spectral counting (Old et al. (2005) Molecular and Cellular Proteomics 4.10. 1487-502) that added a 1.25 correction factor value to all numbers, in order to eliminate any zero values (non detectable values). The algorithm R ₁ , = Log, ₀ (Px + 1.25)/( PO + 1.25)+ Logio (TPO - PO + 1.25)/(TPx - Px +1.25))and R ₄₀ = Logio (SCx + 1.25)/(SC0 + SCx +1.25) + Logio (TSCO - SCO +1.25)/( TSCx - SCx +1.25), where R _p is the Logto ratio of number of unique peptides between time points 0 and x, Rs ₀ is the Logio ratio of spectral counts between time points 0 and x, PO or Px is the number of unique peptides or at baseline (0) or another time point (x) for the specific protein of interest. TPO or TPx is the number of all unique peptides for the complete data set for that individual at that specific time point (0 or x). SCO and SCx are the spectral counts at time points 0 and x for the protein. TSCO and TSCx are the total number of spectral counts in the

experiment at time points 0 and x. There is a linear correlation between number of peptides observed and the spectra count (under 70 peptides/protein). However, for very abundant protein with lOO's of peptides and observations the relationship is more non-linear. A cut off of 0.3 (2 fold) was used to indicate a change.

3. Duplicate MS analysis

Two independent MS analyses were done on each LC fraction. In both cases, LC fractions were stored (-8O ⁰ C) following an independent LC separation of the intact proteins. Image analysis was carried out between the various LC runs to match the fractions as closely as possible. However, the fractions analyzed were not completely identical due to variation in the LC run and exact timing of the fraction collection. The stored fractions were dried down, neutralized and digested with trypsin. The digested sample was applied to the LC LTQ-Orbitrap MS and number of peptides and spectra count were determined (table 5). The total number of peptide and counts for each time points is shown in table 6. This is taken into account when calculating change.

This space intentionally left blank.

'Jl

K>

4. Criteria selection for IDLC

Selection criteria for IDLC was based on analysis of all individuals in each group (induced necrosis or induced ischemia) and are as follows: i) Changes in the number of unique peptides or the number of time each peptide was observed regardless of charge state (2+. 3+ or 4+) were compared to the equivalent protein (if observed) at time point 0. Changes are based on R _p or R ₅₀ values of 0.30 or greater. The fold changes associated with these R values depend on the number of observations and range from 1 :4 ratio (R = 0.36 before total observation correction, 4 fold change) to a 300:600 (R=0.3001 before total observation correction, 2 fold change). The total observation correction will shift the R value depending on the different in size of the 2 sample groups and the proportion of observations for a the protein of interest relative to the entire sample. A 10% difference in sample size between the compared samples could increase or decrease the R value by 0.045U) ii) Changes in 2 out of the 3 or 3 out of the 3 individuals in a designated group (induced ischemia or induced necrosis) at any time point.

5. Results

Although the number of unique peptides observed between MS run 1 and 2 were similar, the number of peptide observations was reduced in the second run (Table 6). This did not overly affect the number of proteins observed except for the lower abundant proteins. There were 25 different serum samples analyzed, giving raise to 650 fractions being analyzed per MS analysis (1300 total MS runs). This resulted in, for both MS runs, over 41,000 unique peptides being identified, with these peptides observed over 410,600 times. Table 7 outlines the proteins that met the criteria above and whether they were observed to change by 2DLC.

An additional level of stringency was added in which the variation within an individual was taken into account. The first tier proteins are found consistently to remain elevated. These are highlighted in bold type. Those proteins with variation between patients or time points were ranked as second tier. Bradykinin peptide was also included (rather than kininogen, the parent protein which had a weaker correlation) based on reducing the fold change required to 1.5 fold. It was the only additional protein that met this weaker criterion. For details regarding the proteins see Example II.

Table 7: summary of protein Protein change changes observed by 1DLC up in detected by protein name accession up in ischemia necrosis 2DLC

2 or 3 of 3 2 or 3 of 3

Alpha-2-HS-glycoprotein precursor IPI00022431 yes Angiogβnin IPI00008554 yes

Caitoxypeptidase N catalytic chain IPI00010295 yes Extracellular matrix protein 1 IPI00645849 yes Galectin-7 IPI00219221 yes Hornerin IPI00398625 ' yes lsoform 1 of Long palate, lung and nasal epithelium carcinoma- associated protein 1 IPI00291410 yes lsoform 2 of Semβnogelin-1 IPI00414684 yes Lumican IPI00020986 yes Profilaggrin IPI00654788 yes

Thyroxine-binding globulin IPI00292946 yes yes Vitamin D-binding protein IPI00555812- yes lsoform LMW of Kininogen-1 (specifically bradykinin) IPI00215894 yes C4b-biπding protein alpha chain IPI00021727 yes yes Proteoglycan-4 (isoforms A and D) IPI00655676/ yes

E. 2-dimensional liquid chromatography 1. 2DLC cohort

Since 2DLC requires 2 mg of protein or greater for each analysis and the quantity of sample for the atria pacing cohort was limited so pooling was required for each group as outlined in table 8.

2.2DLC Method

Serum was depleted using an affinity chromatography comprised of IgY antibodies specific for 3 major forms of immunoglobins (IgG, IgA and IgM) and then depleted of albumin using our in-house affinity /chemical depletion method. This was done in order to reduce background of the chromatogram. Protein concentration was determined using BCA assay (Pierce). Samples were analyzed on the same set of first and second dimension columns. 2mg of sample from each time point were sequentially injected on the HPCF first dimension column.

followed by HPRF second dimension separation. Fractions were collected into 96-well plates and stored at -80C until analyzed. Extensive washing was carried out between runs to eliminate possible cross-over contamination.

Chromatographic images were compared and the same regions/domains from multiple- pH fractions were combined based on profile and previous identification. Fractions from high salt wash were analyzed individually without pooling. Total 315 fractions from 7 time points were dried down, neutralized, and digested with trypsin. 50% of the digested sample was applied to the LC LTQ-Orbitrap MS.

For LC-MS/MS experiments on the LTQ-Orbitrap (ThermoFinnigan, San Jose, CA), peptides from the digestion of LC fraction were resuspended in 6 μl resuspension buffer (4% acetonitrile in water with 0.1% formic acid). Samples (3 μl) were loaded onto a 75um x 10 cm BioBasic Cl 8 column (New Objective, Woburn, MA). Peptides were cluted into an LTQ- Orbitrap (ThermoFinnigan, San Jose, CA) using an Agilent 1200 nano-LC system (Agilent, Santa Clara, CA). The HPLC gradient was 5% to 60% B (90% acetonitrile/watcr in 0.1% Formic acid) over 30 or 60 min dependent on sample complexity. The mass spectrometer was operated in data-dependent mode in which every FT-MS scan (survey 350-2000 Da) was followed by MS/MS scans of the 5 most abundant ions.

All mass spectrometry data was analyzed according to the pipeline established and already used in the Van Eyk laboratory, designed to meet stringent criteria from the proteomics community. Data from the LTQ-Orbitrap was searched against the IPI databases where possible, using Sorcerer Sequest (Sagen, San Jose, CA). Search results were validated and analyzed using Scaffold (Proteome Systems, Portland, OR). Protein identifications based upon a single peptide observation were handled carefully through manual inspection of the tandem MS (MS/MS) spectra, BLAST searching of the sequence to ensure it matches only the reported protein, requiring a minimum of 8 amino acids and a peptide probability score of >0.9.

Data analysis flow was based on the following: peptide redundancy removed, protein name redundancy removed, the number of unique peptides and number of observation for each peptide regardless of charge state were determined for each protein and, in the cases where the protein was observed in multiple domains as an indicator of potential PTM this was noted and all data for the protein was included for quantitation.

The number of unique peptides and total number of counts (number of times each peptide is observed) were dependently used to semi-quantify each protein that was observed. To deal with proteins which peptides were only observed at some but not all time points (resulting

in no information) a correction factor of 1.25 was used in all R value calculations with a R > 0.3 as indicative of change. See IDLC for more detail.

3. Criteria selection for 2DLC

A) Individual in each group (induced necrosis or induced ischemia) i) Changes in the number of unique peptides or the number of times each peptide- was observed regardless of charge state (+2. +3 or +4) were compared to the equivalent protein (if observed) at time point 0. All proteins only seen in time points after baseline were included. ii) Changes in 3 out of the 3 or 2 out of the 3 individuals in a designated group (induced ischemia or induced necrosis) at any time point.

B) Group i) proteins that met the above criteria for either induced ischemia or induced necrosis that did not change in all of the control (non ischemic) samples at any time point.

4. 2DLC Results

Over 7500 specta were analyzed per pooled sample and thus, over 52,5000 spectra in total. Spectra were removed based on poor quality and each mass was assigned to a single peptide sequence resulting in ~55OO spectra remaining. ITie number of the proteins that met the criteria are listed below in table 9. See Example II for detailed protein information.

Table 9 : protein observed changed by 2OLC

IPI accession

Protein name number increased increased potential PTM present at Ischemia Ischemia present at necrosis Control Control_ present at in Ischemia in necrosis in any sample baseline T1/T0 T2/T0 baseline T1/T0 baseline T1/T0

Protease, seπne, 3 IPI00748381 yes yes no O O > O > O O

C4b-bindιπg protein alpha chain IPI00021727 yes yes yes yes O > yes > yes

Alpha-2-glycoproteιπ 1, zinc (no) IPI00166729 yes yes yes O > > O > O O

Thyroxiπe-blnding globulin precursor IPI00292946 no yes no yes O O O > O O

Criteria was that both number of peptide and number of count must increased increased in ischemia = if protein is Increases over baseline in the PCOLEO sample comprising severe ischemia patients. Ischemia was based on increase in lactate over time and no detectable cTnT increased in necrosis = if protein is increases over baseline in the POOLED sample compnsing necrosis patients analyzed Necrosis was based on detectable cTnT (at time point 2 and or 3)

PTM = protein found in multiple fractions in any of the pooled sample. Note, if sequential maybe reflect not PTM but rather large quantity of protein elutlng over multiple fractions yes= present at baseline O not detected, > greater than baseline < less than baseline = equal to baseline yes - detected at baseline control pool' all TO = baseline from 13, 19, 9, T1= time point 1 for 13,19 and 9 ischemia pool: TO = baseline from 7,10,11; T1 = time point 1 from 7,10,11; T2 = time point 2 and 3 from 7, 10,11 necrosis pool TO = baseline and time point 1 for 16,15; T2= tone points 2 and 3 of patient 16 and 15

Interestingly, two proteins, junction plakoglobin, desmoplakin, that are part of the desmonsomal protein complex were detected in a few patients but not enough to make it meet the criteria. Desmoglein-1, the other protein of the same complex was found elevated in necrosis individual with 2DE.

Increase in Ischemia Ischemia Ischemia θ IPI # Ischemia TP1 TP2 TP3

;tion plakoglobin snin gamma Isαform 1) IPI00554711 yes 0 0 isoform (most likely IPI00217182

DPI) Desmoplakin (IPI0013933) yes 0 0

F. Final summary of all protein changes with three methods of proteomic analysis

Coronary sinus serum samples were analyzed from patients undergoing atrial pacing. Patients were designated into control, ischemia or necrosis groups based on the presence of cTnT at any time point (necrosis group) and an increase in lactate during the pacing protocol (ischemia and necrosis groups). Depleted samples were analyzed for all patients and all time points by 2DE. Depleted samples for 3 patients that were control, ischemia or necrosis were analyzed at multiple time points by 1 DLC. Pooling of these individuals (2 or 3) from control, necrosis and ischemia were required for analyzed by 2DLC due to the amount of protein required for this technology. Proteins selection criteria for each method and MS-based quantitation for each method are described above.

The following proteins have been selected as primary or secondary targets based on the robustness of their changes with ischemia and or necrosis and known biological functions. The overlap is schematically shown in figure 5. Detailed information about each target is located in Example IT.

Primary targets

Lumican

Extracellular matrix protein 1

Angiogenin

Semenogelin (all isoforms 1 and 2)

Long palate, lung and nasal epithelium carcinoma-associated protein 1 (all isoforms, 1 and 2)

Secondary targets

Alpha-2-HS-glycoprotein

Carboxypeptidase N (all subunits including catalytic chain)

Galectin-7

Hornerin

Proteoglycan-4 (Isoform A and D)

Profilaggrin (Filaggrin)

Vitamin D binding protein

C4b-binding protein alpha chain

Thyroxine binding globulin

λlpha-2-glycoprotein 1, zinc protease, serine 3

Caspase 14

Desmogelin

Kininogen - 1 (LMW and HMW and bradykinin)

Example II. Summary of some of the properties of proteins discussed with regard to cohort I

A. Vitamin D-binding protein

Name: Vitamin D-binding protein

IPI ID: IPI00555812

UniProtKB/Swiss-Prot entry ID: P02774, Ol 6309, 016310 Length: 474 aa, molecular weight: 52964 Da (of precursor)

1. Basic Information from UniProtKB/Swiss-Prot entry

• FUNCTION: Multifunctional protein found in plasma, ascitic fluid, cerebrospinal fluid, and urine and on the surface of many cell types. In plasma, it carries the vitamin D sterols and prevents polymerization of actin by binding its monomers. DBP associates with membrane-bound immunoglobulin on the surface of B-lymphocytes and with IgG Fc receptor on the membranes of T-lymphocytcs.

• SUBCFXLULAR LOCATION: Secreted.

• POLYMORPHISM: Over 80 variants of human DBP have been identified. The three most common alleles are called GC* IF, GC* IS, and GC*2. The sequence shown is that ofthe GC*2 allele

2. Sequence

MKi?VLVLILAraFGHALERGRDYEKNKVCKEFSHLGKEDFTSLSLVLYSRKFPSGT FEQV SQLVK EWSLTEACCAEGADPDCYDTRTSALSAKSCESNSPFPVHPGTAECCTKEGLERK LCMAALKHQPQEFPTYVEPTNDEICEAFRKDPKEYANQFMWEYSTNYGQAPLSLLVSYTK SYLSMVGSCCTSASPTVCFLKERLQLKHLSLLTTLSNRVCSQYAAYGEKKSRLSNLIKLA QKVPTADLEDVLPLAEDITNILSKCCESASEDCMAKELPEHTVKLCDNLSTKNSKFEDCC QEKTAMDVFVCTYFMPAAQLPELPDVELPTNKDVCDPGNTKVMDKYTFELSRRTHLPEVF LSKVLE PTLKS LGECCDVEDSTTCFNAKGPLLKKELSSFIDKGQELCADYSENTFTEYKK KLAERLKAKLPDATPKELAKLVNKRSDFASNCCSINSPPLYCDSEIDAELKNIL (SEQ ID NO : 1 )

1-16 leader sequence .

Peptides used in the MS analysis described in this application are indicated by highlighting (shading) in the protein sequences shown herein. A skilled worker can use peptides for some of the proteins which have been described previously by others who have performed MS on those proteins. The sequences of peptides is dependent on the particular type of MS used. For example, the peptides for MALDI can be different from those in ESI. The studies performed herein were ESI.

3. Alternative names: DBP, Group- specific component, Gc-globulin, VDB

4. Additional information on function

• Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF).

• Gc protein was deglycosylated by serum alpha-N-acelylgalactosaminidase (Nagalase)

• The level of Gc globulin is reduced in patients with fulminate hepatic failure, septic shock and trauma. Furthermore, low levels of Gc globulin in patients with fulminant hepatic failure and multiple trauma have been found to correlate with the morbidity and mortality of patients. It has not been studied in heart disease.

5. Summary

Assays are available for total Vitamin D binding protein and for the amount of protein which is either free or bound to actin. This protein is known to be diagnostic for several diseases. It seems to be change with cellular injury and decrease with long term chronic disease. Clinical studies and animal models have shown that Gc-globulin has an important role in the clearance of procoagulant actin from the circulation after its release during cell necrosis and tissue injury but it is not known if it is in the heart.

B. Thyroxine-hindinp globulin

Name: Thyroxine-binding globulin precursor

IPl ID: IPI00292946

UniProtKB/Swiss-Prot entry ID: P05543 Length: 415 aa, molecular weight: 46325 Da (precursor)

1. Basic Information from UniProtKB/Swiss-Prot entry

• FUNCTION: Major thyroid hormone transport protein in serum. . SUBCELLULAR LOCATION: Secreted.

• TISSUE SPECIFICITY: Expressed by the liver and secreted in plasma.

• DISEASE: Defects in SERPINA7 are a cause of TBG deficiency γM1M:3142001. Mutations in the SERPIN A7 gene can result as a whole spectrum of deficiencies, characterized by either reduced or increased TBG levels in the serum. Patients show, respectively, reduced or elevated protein-bound iodine but are euthyroid.

2. Sequence

MSPFL YL VLL VLGLHAγIHCASPEGKVTACHSSQPNATLYKMSSINAUFAFNLYRRFTVE

TPDKNIFFSPVSISAALVMLSFGACCSTQTEIVETLGFNLTDTPMVEIQHGFQHLIC SLN

FPKKELBLQIGNALFIGKHLKPIAKFLNDVKTLYETEVFSTDFSNISAAKQEINSHV EMQ

TKGKWGLIQDLKPNTIMVLVNYIHFKAQWANPFDPSKTEDSSSFLIDKTTTVQVPMM HQ

MEQYYHLVDMELNCTVLQMDYSKNALALFVLPKEGQMESVEAAMSSKTLKKWNRLLQ KGW

VDLFVPKFSISATYDLGATLLKMGIQHAYSENADFSGLTEDNGLKLSNAAHKAVLHI GEK

GTEAAAVPEVELSDQPENTFLHPIIQIDRSFMLLILERSTRSILFLGKWNPTEA (SEQ ID

NO : 2 )

1-20 signal sequence

3. Alternative names: T4-binding globulin, Serpin A7

4. Summary

Thyroxine-binding globulin binds with high-affinity to the thyroid hormone. It is proposed to be a biomarker for senescence and aging. Chronic treatment with perindopril, an angiotensin I- converting enzyme inhibitor used in cardiac and renal disease, enhanced thyroxine-binding capacity and possibility the protein level itself. In a study on ACS, thyroxine binding globulin was measured in those with acute myocardial infarction after 14 days and there was no change compared to control. It has not been studied in myocardial ischemia or events leding up to MI.

C Lumican

Name: Lumican

IPI ID: IPI00020986

UniProtKB/Swiss-Prot entry ID:

Length: 338 aa, molecular weight: 38429 Da, (of precursor)

1. Basic Information from UniProtKB/Swiss-Prot entry

SUBUNIT Binds to laminin (By similarity).

SUBCELLULAR Secreted, extracellular space, extracellular matrix (By

LOCATION similarity).

2. Sequence

MSLSAFTLFLALIGGγSGQYYDYDFPLSIYGQSSPNCAPECNCPESYPSAMYCDξ LKLKS

VPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGRVFSKLKQ LKK LHINHNNLTESVGPLPKSLEDLQLTHNKITKLGSFEGLVNLTFIHLQHNRLKEDAVSAAF KGLKSLEYLDLSFNQIARLPSGLPVSLLTLYLDNNKISNIPDEYFKRFNALQYLRLSHNE LADSGIPGNSFNVSSLVELDLSYNKLKNIPTVNENLENYYLEVNQLEKFDIKSFCKILGP LSYSKIKHLRLDGNRISETSLPPDMYECLRVANEVTLN (SEQ ID NO: 3)

1-18 signal sequence

3. Alternative names:

(Keratan sulfate proteoglycan lumican) (KSPG lumican).

4. Summary

Protein involved in injury response in a number of tissues and is a secreted protein. Tn a study on the lumican in fibrosis with chronic ischemic and reperfused rat heart (which is not the acute myocardial infaction model, but rather would induce heart failure), the level of lumican mRNA increased, peaking at the fourth week. The protein level was not investigated. This protein is also known to inhibits cell adhesion and neurite outgrowth and be involved in wound healing of the cornea. It plays an important role in cell migration and proliferation during embryonic development, tissue repair, and tumor growth. It has not been studied in the context of myocardial ischemia or events leading up to MI.

D. GaIectin-7

Name: Galectin-7

IPI ID: IPI00219221

UniProtKB/Swiss-Prot entry ID:

Length: 136 aa, molecular weight: 15075 Da

1. Basic Information from UniProtKB/Swiss-Prot entry

Could be involved in cell-cell and/or cell-matrix interactions necessary FUNCTION for normal growth control. Pro-apoptotic protein that functions intracellularly upstream of .INK activation and cytochrome c release.

SUBCELLULAR Cytoplasm. Nucleus. Secreted (Potential). Note=May be secreted by a LOCATION non-classical secretory pathway.

TISSUE Mainly in stratified squamous epithelium.

SPECIFICITY

2. Sequence

WSNVPHKSSLPEGIRPGTVLRIRGLVPPNASRFHVNLLCGEEQGSDAALHFNPR]LD TSEV

VFNSKEQGSWGREERGPGVPFQRGQPFEVLIIASDDGFKA WGDAQYHHFRHRLPLARVR LVEVGGDVQLDSVRIF (SEQ ID NO : 4 )

Initial met is removed

3. Alternative names: Galectin-7 (Gal-7) (HKL- 14) (PI7) (p53-induced gene 1 protein).

Homologous 100% to O6IB87 HUMAN LGALS7 protein (HCG1776519) (HCG42850) [LGALS7]

Note on sequence: Note only 36 % homology with galectin 3 which is known to be involved in cancer.

4. Additional information on function

The literature suggests that galectin 7 is involved in apoptosis. It is most likely is secreted and forms dimers. Galectin 7 is an emerging marker involved in the epidermal development of pluristratified epithelia and in epidermal cell migration. It is elevated in wound healing. It has not been studied in the context of myocardial ischemia or events leading up to MI.

£• Intracellular matrix protein 1

Name: Extracellular matrix protein 1 IPI ID: IPI00645849

UniProtKB/Swiss-Prot entry ID: O5T5G4

Length: 567 AA (includes signal sequence)

Molecular weight: 63563 Da (includes signal sequence) 1. Basic Information from UniProtKB/Swiss-Prot entry

. FUNCTION: Not known

• SUBCELLULAR LOCATION: Secreted, extracellular space.

• DISEASE: Defects in ECMl are the cause of lipoid proteinosis (LiP); also known as lipoid proteinosis of Urbach and Wiethe or hyalinosis cutis et mucosae. LiP is a rare autosomal recessive disorder characterized by generalized thickening of skin, mucosae and certain viscera. Classical features include beaded eyelid papules and laryngeal

infiltration leading to hoarseness. Histologically, there is widespread deposition of hyaline material and disruption/reduplication of basement membrane.

2. Sequence information from IDLC

MGr2αRAλLViryLAyA5AASEGGFTATGQRQLRPEHFQEVGYAAPPSPPLSRSL PMDHPD

SSQHGPPFEGQSGKEGRGPRPHSQPWLGERVGCSHIPPSIVQPPPSQEATPLQQEKL LPAQ

LPAEKEVGPPLPQEAVPLQKELPSLQHPNEQKEGTPAPFGDQSHPEPESWNAAQHCQ QDRS

QGGWGHRLDGFPPGRPSPDNLNQICLPNRQHWYGPWNLPQSSYSHLTRQGETLNFLE IGY

SRCCHCRSHTNRLECAKLVWEEAMSRFCEAEFSVKTRPHWCCTRQGEARFSCFQEEA PQPH

YQLRACPSHQPDISSGLELPFPPGVPTLDNIKNICHLRRFRSVPRNLPATDPLQREL LALI

QLEREFQRCCRQGNNHTCTWKAWEDTLDKYCDREYAVKTHHHLCCRHPPSPTRDECF ARRA

PYPNYDRDILTIDIGRVTPNLMGHLCGNQRVLTKHKHIPGLIHNMTARCCDLPFPEQ ACCA

EEEKLTFINDLCGPRRNIWRDPALCCYLSPGDEQWCFNINYLRNVALVSGDTENAKG QGE.

QGSTGGTNISSTSEPKEE (SEQ ID NO: 5)

3. Alternative names: Secretory component p85; 05TSGS, O81Z60. Q5T5G6.016610

Note on sequence:

Signal sequence 1-19 is removed in ther mature protein.

4. Summary

Mutations of this protein result in lipoid proteinosis, a rare recessive disorder of the skin and mucosae. It binds perlecan, MMP9 and fibulin in the skin. It can inhibit MMP9. Auto antibodies to this protein occur with lichen sclerosus. Neither disease is common and so specificity to ischemia is likely. It has not been studied in the context of myocardial ischemia or events leading up to MI.

F. Semenogelin _1

Name: Semenogelin 1

IPI ID: IPI00414684 (Semenogelin-2 precursor IPI00025415)

UniProtKB/Swiss-Prot entry ID: P04279. 06X419. O6Y809. O6Y822. O6Y823. O86U64.

O96OM3

Length: 402 aa, molecular weight: 45322 Da

1. Basic Information from UniProtKB/Swiss-Prot entry

Predominant protein in semen. It participates in the formation of a gel matrix entrapping the accessory gland secretions and ejaculated

FUNCTION spermatozoa. Fragments of semenogelin and/or fragments of the related proteins may contribute to the activation of progressive sperm movements as the gel-forming proteins are fragmented by KLK3/PSA.

Alpha-inhibin-92 and alpha-inhibin-31, derived from the proteolytic FUNCTION degradation of semenogelin, inhibit the secretion of pituitary follicle- stimulating hormone.

_ST FRT _JN TT Occurs in disulfide-linked complexes which may also contain two less abundant 71- and 76-kDa semenogelin-related polypeptides.

SUBCELLULAR Secreted. LOCATION

Event=Altemative splicing; Named isoforms=2;

Name=l;

ALTERNATIVE IsoId=P04279-l; Sequence=Displayed; PRODUCTS Name=2;

IsoId=P04279-2; Sequence=VSPJ)04385;

Note=No experimental confirmation available;

TISSUE Seminal vesicle. However references show it is also present in other

SPECIFICITY tissues including skeletal muscle

2. Sequence

MKPNIIFVLSLLLILEKQAAVMGQKGGSKGRLPSEFSQFPHGQKGQHYSGQKGKQQT ESK GSFSIQYTYHVDANDHDQSRKSQQYDLNALHKTTKSQRHLGGSQQLLHNKQEGRDHDKSK GHFHRWIHHKGGKAHRGTQNPSQDQGNSPSGKGISSQYSNTEERLWVHGLSKEQTSVSG AQKGRKQGGSQSSYVLQTEELVANKQQRETKNSHQNKGHYQNWEVREEHSSKVQTSLCP AHQDKLQHGSKDIFSTQDELLVYNKNQHQTKNLNQDQQHGRKANKISYQSSSTEERRLHY GENGVQKDVSQRSIYSQTEKLVAGKSQIQAPNPKQEPWHGENAKGESGQSTNREQDLLSH EQKGRHQHGSHGGLDIVIIEQEDDSDRHLAQHLNNDRNPLFT (SEQ ID NO: 6)

1-23 signal sequence

3. Alternative names:

SEMG

4. Summary

SGI isoform is found in skeletal muscle as well as epithelial cells. Isoform expression is tissue specific and SGI isoform is found in skeletal muscle as well as epithelial cells. The peptides produced by cleavage of semenogelin I, the predominant human semen coagulum protein, had high levels of antibacterial activity. It has not been studied in the context of myocardial ischemia or events leading up to MI.

G. Isoform 1 of Long palate,, lung and nasal epithelium carcinoma-associated protein 1

Name: Isoform 1 of Long palate, lung and nasal epithelium carcinoma-associated protein I IPI ID: IPI00291410.

UniProtKB/Swiss-Prot entry ID: O8TDL5-1 Length: 484 aa, molecular weight: 52442 Da

1. Basic Information from UniProtKB/Swiss-Prot entry

FUNCTION May play a role in innate immunity in mouth, nose and lungs.

SUBCELLULAR Secreted (By similarity).

LOCATION

Event=Alternative splicing; Named isoforms=2;

Name=l;

ALTERNATIVE IsoId=Q8TDL5-l; Sequence=Disρlayed;

PROTYπPT^ Name— ^λ , υ . lsoId=Q8TDL5-2; Sequence=VSP_015285, VSPJ) 15286,

VSPJ) 15287,

VSP 015288;

Note=No experimental confirmation available; ηSSUE SPECIFICITY Detected in trachea, nasal septal epithelium and lung.

O hits

2. Sequence

MAGPWTFTLLCGLLAATLIQATLSPTAVLILGPKVIKEKLTQELKDHNATSILQQLP LLS AMREKPAGGIPVLGSLVNTVLKHIIWLKVITANILQLQVKPSANDQELLVKIPLDMVAGF NTPLVKTIVEFHMTTEAQATIRMDTSASGPTRLVLSDCATSHGSLRIQLLHKLSFLVNAL AKQVMNLLVPSLPNLVKNQLCPVIEASFNGMYADLLQLVKVPISLSIDRLEFDLLYPAIK GDTIQLYLGAKLLDSQGKVTKWFNNSAASLTMPTLDNIPFSLIVSQDWKAAVAAVLSPE EFMVLLDSVLPESAHRLKSSIGLINEKAADKLGSTQIVKILTQDTPEFFIDQGHAKVAQL IVLEVFPSSEALRPLFTLGIEASSEAQFYTKGDQLILNLNNISSDRIQLMNSGIGWFQPD VLKNIITEIIHSILLPNQNGKLRSGVPVSLVKALGFEAAESSLTKDALVLTPASLWKPSS PVSQ (SEQ ID NO: 7)

1-21 potential signal sequence. Isoform 2 is truncated at N-terminus. Also have insert (underlined) which has no peptides. Therefore, cannot distinguish between isoform one and two

3. Alternative names: C20orfl 14

4. Summary

Little is known about this protein or its shorter isoform (2). It has not been studied in the context of myocardial ischemia or events leading up to MI.

JEL-Angiflgfinin

Name: ^• Angiogenin, precursor

IPI ID: IPI00008554

UniProtKB/Swiss-Prot entry ID: P03950

Length: 147 AA [This is the length of the unprocessed precursor]

Molecular weight: 42051 Da [This is the MW of the unprocessed precursor

1. Basic Information from UniProtKB/Swiss-Prot entry

• FUNCTION: May function as a tRNA-specific ribonuclease that binds to actin on the surface of endothelial cells; once bound, angiogenin is endocytosed and translocated to the nucleus, thereby promoting the endothelial invasiveness necessary for blood vessel formation. Angiogenin induces vascularization of normal and malignant tissues. Abolishes protein synthesis by specifically hydrolyzing cellular tRNAs.

• INTERACTIONS: May bind alpha-actinin P35609

• SUBCELLULAR LOCATION: Secreted

• TISSUE SPECIFICITY: Expressed predominantly in the liver.

• DEVELOPMENTAL STAGE: Low level expression in the developing fetus, increased in the neonate, and maximal in the adult

• SIMILARITY: Belongs to the pancreatic ribonuclease family.

It is uncertain whether Met- 1 or Met-3 is the initiator.

2. Sequence information from IDLC

MVMGLGVLLL VFVLGLGLTP PfLAQDNSRY THFLTQHYDA KPQGRDDRYC ESIMRRRGLT SPCKDINTFI HGNKRSIKAI CENKNGNPHR ENLRISKSSF QVTTCKLHGG SPWPPCQYRA TAGFRNVWA CENGLPVHLD QSIFRRP (SEQ ID NO: 8)

Multiple peptides

3. Alternative names:RNASE5, Ribonuclease A family, 5, RNASE4 protein, ANG protein

053X86 . 06P5T2

Note on sequence:

• Most likely have either intact molecule or the mature processed form.

• Signal peptide residues 1-24

Additional information on function Angiogenin is a normal constituent of the circulation and contained in a vasculature that rarely undergoes proliferation, but in some physiological and pathological conditions its levels increase in blood, promoting neovascularization. This is a potentially important physiological protein involved in angiogenesis.

4. Summary

Interestingly, this protein may play a role in angiogenesis. Recently it has been potentially linked to poor outcome in ACS patients, which is a chronic condition that can result from many different etiologies. Plasma angiogenin levels was increased in ACS also with ischemic brain damage. However, it has not been studied in the context of myocardial ischemia or events leading up to MI.

I. C4b-binding protein

Name: ' C4b-binding protein (alpha chain)r

IPI ID: IPI00021727

UniProtKB/Swiss-Prot entry ID: P04003

Length: 597 AA [This is the length of the unprocessed precursor]

Molecular weight: 67033 Da [This is the MW of the unprocessed precursor]

1. Basic Information from UniProtKB/Swiss-Prot entry

• FUNCTION: Controls the classical pathway of complement activation. It binds as a cofactor to C3b/C4b inactivator (C3bINA), which then hydrolyzes the complement fragment C4b. It also accelerates the degradation of the C4bC2a complex (C3 convertase) by dissociating the complement fragment C2a. Alpha chain binds C4b. It interacts also with anticoagulant protein S and with serum amyloid P component.

• SUBUNIT: Disulfide-linked complex of alpha and beta chains of 3 possible sorts: a 570 kDa complex of 7 alpha chains and 1 beta chain, a 530 kDa homoheptamer of alpha chains or a 500 kDa complex of 6 alpha chains and 1 beta chain. The central body of the alpha chain homopolymer supports tentacles, each with the binding site for C4b at the end.

• SUBCELLULAR LOCATION: Secreted

• TISSUE SPECIFICITY: Chylomicrons in the plasma.

It is uncertain whether Met-1 or Met- 17 is the initiator Additional information

CRP binds C4b binding proteins and regulations it inhibition of complement system ( Regulation of Complement Activation by C-Reactive Protein: Targeting of the Inhibitory Activity of C4b-Binding Protein ¹ AP Sjδberg et al., J Immuno. 2006, 176: 7612-7620).

C4b-binding protein (C4BP), binds strongly to necrotic cells, irrespective of the cell type used or the method of induction. (C4b-binding protein binds to necrotic cells and DNA, limiting DNA release and inhibiting complement activation L A. Trouw et al., JEM, 2005, 201 , 1937-1948)

2. Sequence

MHPPKTPSGA LHRKRKMAAW PFSRLWKVSD PILFQMTLIA ALLPAVLGNC GPPPTLSI BMWmmB^ FKTGTTLKYT CLPGYVRSHS TQTLTCNSDG EWVYNTFCIY KRCRHPGELR NGQVEIKTDL SFGSQIEFSC SEGFFLIGST TSRCEVQDRG VGWSHPLPQC EIVKCKPPPD IRNGRHSGEE NFYAYGFSVT YSCDPRFSLL GHASISCTVE NETIGVWRPS PPTCEKITCR FVLRGSSVIH CDADSKWNPS PPACSPNSCI RCHPGYKPTT DEPTTVICQK NLRWTPYQGC _Y G _D EISFS _C H ETSRFSAICQ GDGTWSPRTP IYECDKKP—HWτ,. ^q r. ^qy SHWSAPAPQC

KALCRI§1UU3 UHLSVDKDO YVEPENVTIQ CDSGYGWGP QSITCSGNRT WYPEVPKCEW

ETPEGCEQVL TGKRLMQCLP NPEaV/KM-^aEmX^iMfgyBEeESMgBgaiaDSARO STLDKEL (SEQ ID NO : 9 )

Green both IDLC and 2DLC Blue only 2DLC

3. Alternative names: IC4b binding protein, C4b binding protein alpha chain, C4b receptor, C4bP. C4bPA, C4bPALl, Complement component 4 binding protein, alpha like 1, PRP ₅ Proline rich protein Complement component 4 binding protein, alpha. O5VVQ8

Note on sequence:

Most likely intact protein. Several patients had protein present in multiple fractions and although sequential appears to be due to PTM rather than bleed over between two fractions. E do not know what the PTM is at this time. This protein has not been studied in the context of myocardial ischemia or events leading up to MI.

J. Carbffi&yjgnlitlase N catalytic chain

Name: Carboxypeptidase N catalytic chain precursor

IPI ID: IPI00021439

UniProtKB/Swiss-Prot entry ID: P15169

Length: 458 AA [This is the length of the unprocessed precursor]

Molecular weight: 52286 Da [This is the MW of the unprocessed precursor]

1. Basic Information from UniProtKB/Swiss-Prot entry

• FUNCTION: Protects the body from potent vasoactive and inflammatory peptides containing C-terminal Arg or Lys (such as kinins or anaphylatoxins) which are released into the circulation. Note cleaves bradykinin!

• CATALYTIC ACTIVITY: Release of a C-terminal basic amino acid, preferentially lysine.

• SUBUNIT: Tetramer of two catalytic chains and two glycosylated inactive chains.

• SU BCELLULAR LOCATION : Secreted / extracellular space

• SIMILARITY: Belongs to the peptidr - - ^* " ^* - _lm ii _y

2. Sequence

MSDLLSVFLH LLLLFKLVAP VTFRHHRYDD LVRTLYKVQN ECPGITRVYS IGRSVEGRHL YVLEFSDHPG IHEPLEPEVK YVGNMHGNEA LGRELMLQLS EFLCEEFRNR NQRIVQLIQD TRIHILPSMN PDGYEVAAAQ GPNKPGYLVG RNNANGVPLN RNFPDLNTYI YYNEKYGGPN HHLPLPDNWK SQVEPETRAV IRWMHSFNFV LSANLHGGAV VANYPYDKSF EHRVRGVRRT ASTPTPDDKL FQKLAKVYSY AHGWMFQGWN CGDYFPDGIT NGASWYSLSK GMQDFNYLHT NCFEITLELS CDKFPPξEEL QREWLGNREA LIQFLEQVHQ GIKGMVLDEN YNNLANAVIS VSGINHDVTS GDHGDYFRLL LPGIYTVSAT APGYDPETVT VTVGPAEPTL VNFHLKRSIP QVSPVRRAPS RRHGVRAKVQ PQARKKEMEM RQLQRGPA (SEQ ID NO: 10)

3. Alternative names: CPN, Carboxypeptidase N polypeptide 1, Carboxypeptidase N small subunit; Lysine carboxypeptidase, Arginine carboxypeptidase, Kininase-1, Serum carboxypeptidase NSCPN, Anaphylatoxin inactivator, Plasma carboxypeptidase B Q5T287

Note on sequence: Signal sequence 1-20

4. Summary

Interesting protein, which may alter bradykinin levels and creatine kinase levels. Involved in early inflammatory response. Has been shown to be elevated after AMI based on activity assays. Although it has been shown that there is a high degree of variability in carboxypeptidase N in healthy subjects and does not change with acute myocardial infarction patients but may reach maximum at 48 h after onset of chest pain. It has not been studied in the context of myocardial ischemia or events leading up to MI.

K- ProfUaggri.n/Eilaggiiii

Name: Profilaggrin (Filaggrin)

IPI ID: IPI00746718

UniProtKB/Swiss-Prόt entry ID: P2093Q (note only 70% homology)

Length: 406 IAA

Molecular weight: 435170 Da

1. Basic Information from UniProtKB/Swiss-Prot entry

• FUNCTION: Aggregates keratin intermediate filaments and promotes disulfϊde-bond formation among the intermediate filaments during terminal differentiation of mammalian epidermis

• PTM: Filaggrin is initially synthesized as a large, insoluble, highly phosphorylated precursor containing many tandem copies of 324 AA, which are not separated by "large linker". The precursor is deposited as keratohyalin granules. During terminal differentiation it is dephosphorylated and proteolytically cleaved.

• PTM: Undergoes deimination of some arginine residues (citrullination).

. DISEASE: Defects in FLG are the cause of ichthyosis vulgaris [M I M: 146700]: also known as ichthyosis simplex. The phenotypic characteristics of.ichthyosis vulgaris include palmar, hyperlinearity, keratosis pilaris and a fine scale that is most prominent over the lower abdomen, arms, and legs. Ichthyosis vulgaris is characterized histologically by absent or reduced keratohyalin granules in the epidermis and mild hyperkeratosis. The disease can be associated with frequent asthma, eczema or hay fever. Inheritance is autosomal dominant.

2. Sequence (IPI sequence)

MSTLLENIFAIINLFKQYSKKDKNTDTLSKKELKELLEKEFRQILKNPDDPDMVDVF MDH I-DIDHNKKIDFTEFLLMVFKLAQAYYESTRKENLPISGHKHRKHSHHDKHEDNKQEENK E

NRKRPSSLERRNNRKGNKGRSKSPRETGGKRHESSSEKKERKGYSPTHREEEYGKNH HNS SKKEKNKTENTRLGDNRKRLSERLEEKEDNEEGVYDYENTGRMTQKWIQSGHIATYYTIQ DEAYDTTDSLLEENKIYERSRSSDGKSSSQVNRSRHENTSQVPLQESRTRKRRGSRVSQD RDSEGHSEDSERHSGSASRNHHGSAWEQSRDGSRHPRSHDEDRASHGHSADSSRQSGTRH AETSSRGQTASSHEQARSSPGERHGSGHQQSADSSRHSATGRGQASSAVSDRGHRGSSGS QASDSEGHSENSDTQSVSGHGKAGLRQQSHQESTRGRSGERSGRSGSFIYQVSTHEQSES AHGRTRTSTGRRQGSHHEQARDSSRHSASQEGQDTIRAHPGSRRGGRQGSHHEQSVDRSG HSGSHHSHTTSQGRSDVSRGQSGSRSVSRQTRNEKQSGDGSRHSGSRHHEASSRADSSRH SQVGQGQSSGPRTSRNQGSSVSQDSDSQGHSEDSERRSGSASRNHHGSAQEQSRDGSRHP RSHHEDRAGHGHSAESSRQSGTHHAENSSGGQAASSHEQARSSAGERHGSHHQQSADSSR HSGIGHGQASSAVRDSGHRGSSGSQASDSEGHSEDSDTQSVSAHGQAGPHQQSHQESTRG RSAGRSGRSGSFLYQVSTHEQSESAHGRTRTSTGRRQGSHHEQARDSSRHSASQEGQDTI RGHPGSSRRGRQGSHYEQSVDRSGHSGSHHSHTTSQGRSDASRGQSGSRSASRQTRNDEQ SGDGSRHSWSHHHEASTQADSSRHSQSGQGQSAGPRTSRNQGSSVSQDSDSQGHSEDSER WSGSASRNHRGSAQEQSRDGSRHPTSHHEDRAGHGHSAESSRQSGTHHAENSSGGQAASS HEQARSSAGξRHGSHHQQSADSSRHSGIGHGQASSAVRDSGHRGSSGSQASDSEGHSξ DS DTQSVSAHGQAGPHQQSHQESTRGRSAGRSGRSGSFLYQVSTHEQSESAHGRAGPSTGGR QGSRHEQARDSSRHSASQEGQDTIRGHPGSRRGGRQGSYHEQSVDRSGHSGSHHSHTTSQ GRSDASHGQSGS (SEQ ID NO: 11)

3. Summary Peptides observed are unique to Filaggrin (and not to Ifapsoriasin or Hornerin). This protein has not been studied in the context of myocardial ischemia or events leading up to MI.

L- Proteoglvcan-4

Name: Isoform A and D of Proteoglycan-4 (we cannot distinguish isoforms due to high degree of sequence homology)

IPI ID: IPI00024825 and IPI00655676

UniProtKB/Swiss-Prot entry ID: 092954. O6DNC4. O6DNC5. O6ZMZ5. O9BX49

Length: 1404 aa, molecular weight: 151077 Da 1. Sequence

WAP/KTLPIYLLLLLSVFVIQQVSSQDLSSCAGRCGEGYSRDATCNCDYNCQHYMECCPD F KRVCTAELSCKGRCFESFERGRξCDCDAQCKKYDKCCPDYESFCAEVHNPTSPPSSKKA P PPSGASQTIKSTTKRSPKPPNKKKTKKVIESEEITEEHSVSENQESSSSSSSSSSSSTIR KIKSSKNSAANRELQKKLKVKDNKKNRTKKKPTPKPPWDEAGSGLDNGDFKVTTPDTST TQHNKVSTSPKITTAKPINPRPSLPPNSDTSKETSLTVNKETTVETKETTTTNKQTSTDG KEKTTSAKETQSIEKTSAKDLAPTSKVLAKPTPKAETTTKGPALTTPKEPTPTTPKEPAS TTPKEPTPTTIKSAPTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTKEP APTTTKSAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPTPTTPKEPAPTTKEPAPTTPK EPAPTAPKKPAPTTPKEPAPTTPKEPAPTTTKEPSPTTPKEPAPTTTKSAPTTTKEPAPT TTKSAPTTPKEPSPTTTKEPAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPAPTTTKKP _APTTPKEPAPTT PKETA _PTTPK K _LTPT TP _E K _L AP _{TTPEKPAPTTPEELAPTTPEEPTPTT}

PEEPAPTTPKAAAPNTPKEPAPTTPKEPAPTTPKEPAPTTPKETAPTTPKGTAPTTL KEP APTTPKKPAPKELAPTTTKEPTSTTCDKPAPTTPKGTAPTTPKEPAPTTPKEPAPTTPKG TAPTTLKEPAPTTPKKPAPKELAPTTTKGPTSTTSDKPAPTTPKETAPTTPKEPAPTTPK KPAPTTPETPPPTTSEVSTPTTTKEPTTIHKSPDESTPELSAEPTPKALENSPKEPGVPT TKTPAATKPEMTTTAKDKTTERDLRTTPETTTAAPKMTKETATTTEKTTESKITATTTQV TSTTTQDTTPFKITTLKTTTLAPKVTTTKKTITTTEIMNKPEETAKPKDRATNSKATTPK PQKPTKAPKKPTSTKKPKTMPRVRKPKTTPTPRKMTSTMPELNPTSRIAEAMLQTTTRPN QTPNSKLVEVNPKSEDAGGAEGETPHMLLRPHVFMPEVTPDMDYLPRVPNQGIIINPMLS

DETNICNGKPVDGLTTLRNGTLVAFRGHYFWMLSPFSPPSPARRITEVWGIPSPIDT VFT RCNCEGKTFFFKDSQYWRFTNDIKDAGYPKPIFKGFGGLTGQIVAALSTAKYKNWPESVY FFKRGGSIQQYIYKQEPVQKCPGRRPALNYPVYGETTQVRRRRFERAIGPSQTHTIRIQY SPARLAYQDKGVLHNEVKVSILWRGLPNWTSAISLPNIRKPDGYDYYAFSKDQYYNIDV PSRTARAITTRSGQTLSKVWYNCP (SEQ ID NO: 12)

1 24 24 Potential, signal 25 1404 1380 Proteoglycan-4. 1307 1404 98 Proteoglycan-4 C-terminal part.

26 66 41 Missing (in isoform B, isoform D and isoform E).

107 199 93 Missing (in isoform C and isoform D).

157 199 43 Missing (in isoform F).

412 841 430 Missing (in isoform E).

2. Alternative names: Proteoglycan-4 precursor (Lubricin) (Mcgakaryocyte-stimulating factor) (Superficial zone proteoglycan) [Contains: Proteoglycan-4 C-terminal part].

3. Summary

PRG4 (proteoglycan 4) is a megakaryocyte stimulating factor and articular superficial zone protein which is expressed in cartilage, liver, heart, lung, and bone. It is known to be involved in the lubrication of mammalian joints. This protein has not been studied in the context of myocardial ischemia or events leading up to MI.

IVT. Alpha-2-HS-glycoproleJn

Name: Alpha-2-HS-glycoprotein

IPI ID: , IPI00022431

UniProtKB/Swiss-Prot entry ID: P02765 Length: 367 aa, molecular weight: 39325 Da

1. Basic Information from UniProtKB/Swiss-Prot entry

FT INCTTON Promotes endocytosis, possesses opsonic properties and influences the mineral phase of bone. Shows affinity for calcium and barium ions.

^• Alpha-2-HS glycoprotein derives from this precursor, when the

SUBUNIT connecting peptide is cleaved off. The two chains A and B are held together by a single disulfide bond.

SUBCELLULAR Secreted.

LOCA TION

_F Synthesized in liver and selectively concentrated in bone matrix. Secrete

_CDET ¹ _TFT r _MTV ^din P ^lasma - K ^{is also found in} dentin in much higher quantities than

. other plasma proteins.

2. Sequence i^SLVLLLCLAQLWGCHSAPHGPGLIYRQPNCDDPETEEAALVAIDYINQNLPWGYKHTL NQIDEVKVWPQQPSGELFEIEIDTLETTCHVLDPTPVARCSVRQLKEHAVEGDCDFQLLK LDGKFSWYAKCDSSPDSAEDVRKVCQDCPLLAPLNDTRWHAAKAALAAFNAQNNGSNF QLEEISRAQLVPLPPSTYVEFTVSGTDCVAKEATEAAKCNLLAEKQYGFCKATLSEKLGG AEVAVTCTVFQTQPVTSQPQPEGANEAVPTPWDPDAPPSPPLGAPGLPPAGSPPDSHVL LAAPPGHQLHRAHYDLRHTFMGWSLGSPSGEVSHPRKTRTWQPSVGAAAGPWPPCPG RIRHFKV (SEQ ID NO : 13 )

1-18 signal sequence ^'

3. Alternative names:

Alpha-2-HS-glycoprotein precursor (Fetuin-A) (Alpha-2-Z-globulin) (Ba- alpha-2- glycoprotein) [Contains: Alpha-2-HS-glycoprotein chain A; Alpha-2-HS-glycoprotein chain B].

4. Summary Very high abundant protein and found to change in many diseases and acts as a calcification inhibitor. It inhibits inflammation. It is elevated late after acute myocardial infarction but did not correlate with peak cardiac troponin values. This protein has not been studied in the context of myocardial ischemia or events leading up to MI.

JN. Protease, serine, 3 (mesotrypsinogen) IPI00748381

Name: Protease, serine, 3

IPI ID: IPI00748381

UniProtKB/Swiss-Prot entry ID: 05 JT 15

Length: , 249AA [This is the length of the unprocessed precursor]

Molecular weight: 26914 Da [This is the MW of the unprocessed precursor

1. Basic Information from UniProtKB/Swiss-Prot entry

• FUNCTION fmesotrypsinogen data): Preferential cleavage: Arg-|-Xaa, Lys-|-Xaa. Cofactor Binds 1 calcium ion per subunit.

• INTERACTIONS : This protein binds to Amyloid beta A4 (which we observe but do not see changed) and tissue factor pathway inhibitor (see HPRD)

• SUBCELLULAR LOCATION: Secreted.

• TISSUE SPECIFICITY: Pancreas and brain.

2. Sequence information from 2DLC

Note: this protein was identified in different fractions for ischemia vs AMI. This suggests that this protein has undergone a PTM with AMI and thus is physically distinct from the form present during ischemia. We do not know what this PTM(s) is at this time.

Sequence

MNPFLILAFVGAAGEVAVPFDDDDKIVGGYTCEENSLPYQVSLNSGSHFCGGSLISE QWV VSAAHCYKTRIQVRLGEHNIKVLEGNEQFINAAKIIRHPKYNRDTLDNDIMLIKLSSPAV INARVSTISLPTTPPAAGTECLISGWGNTLSFGADYPDELKCLDAPVLTQAECKASYPGK ITNSMFC^GFLEGGKDSCQRDSGGPVVCNGQI^GVVSWGHGCAWKHRPGVYTKVYNYVDW IKDTIAANS (SEQ ID NO: 14) bold amino acids are trypsin-like domain

3. Alternative names:

Uncharacterized protein PRSS3 A6NN76. Mesotrypsin C O6ISJ4. Mesotrypsinogen C P35O30-3

(98% homologous and not in region of the observed peptides), Isoform C of P35030 P35030-3,

Isoform B of P35030 P35030-2. Based on HPRD this is the same protein as trypsinogen lV(has same sequence), protease serine, 4, TR Y3, TR Y4, trypsin 3, trypsin 4 (Brain), trypsinogen III

(pancreatic).

Note: In swiss prot, mesotrysinogen has three isoforms - two of which are longer proteins at the

N-terminus. We can not distinguish between the three isoforms.

Note on sequence:

We cannot distinguish between the three highly conserved isoforms of mesotrypsinogen based on

MS data.

4. Summary

Mesotrypsin is an inhibitor-resistant protease and is secreted from pancreatic juice. Whether it is present in the heart is not known. This protein has not been studied in the context of myocardial ischemia or events leading up to MI.

Q. Alpha-2-fllγcoprntein l _t zinc IPIOO 166729

Name: alpha-2-glycoprotein 1, zinc

IPI ID: IPI00166729

UniProtKB/Swiss-Prot entry ID: Q8N4N0 O5XKO4

Length: 298 AA [This is the length of the unprocessed precursor]

Molecular weight: 34259 Da [This is the MW of the unprocessed precursor

1. Basic Information from UniProtKB/Swiss-Prot entry

• FUNCTION: Stimulates lipid degradation in adipocytes and causes the extensive fat losses associated with some advanced cancers. May bind polyunsaturated fatty acids.

• SUBCELLULAR LOCATION: Secreted.

• TISSUE SPECIFICITY: Blood plasma, seminal plasma, urine, saliva, sweat, epithelial cells of various human glands, liver.

2. Sequence

MVRMVPVLLSLLLLLGPAVPQENQDGRYSLTYIYTGLSKHVEDVPAFQALGSLNDLQ FFR YNSKDRKSQPMGLWRQVEGMEDWKQDSQLQKAREDIFMETLKDIVEYYNDSNGSHVLQGR FGCEIENNRSSGAFWKYYYDGKDYIEFNKEIPAWVPFDPAAQITKQKWEAEPVYVQRAKA YLEEECPATLRKYLKYSKNILDRQDPPSVWTSHQAPGEKKKLKCLAYDFYPGKIDVHWT RAGEVQEPELRGDVLHNGNGTYQSWWVAVPPQDTAPYSCHVQHSSLAQPLWPWEAS (SEQ ID NO: 15)

Note on sequence: note initiating Met is cleaved. There maybe a PTM with ischemia as the peptides elute at different fractions at T-I and T-2. We do not know what the PTM is.

3. Alternative names: Alpha-2-glycoprotein 1 Q5XKO4, zinc binding; zinc-alpha-2- glycoprotein P25311 (295 AA and 33872 Da, over 95% homology)

4. Summary Zinc-alpha2-glycoprotein (ZAG) is a a lipid mobilizing factor found in adipose tissue. It is increased in a number of cancers. Nothing is known about with respect to the heart and myocardial ischemia. It has not been studied in the context of myocardial ischemia or events leading up to MI.

P. DcSiHOgIcJn-I ₁

Name: Desmoglein-1

IPI ID: I PI00025753

UniProtKB/Swiss-Prot: 002413

Length: 1049 AA [This is the length of the unprocessed precursor]

Molecular Weight: 113716 Da

1. Basic information from UniProtKB/Swiss-Prot entry Q02413

• FUNCTION: Component of intercellular desmosome junctions. Involved in the interaction of plaque proteins and intermediate filaments mediating cell-cell adhesion.

• SUBCELLULAR LOCATION: Cell membrane; Single-pass type I membrane protein (By similarity).

• SIMILARITY: Contains 4 cadherin domains

• TISSUE SPECIFICITY: Epidermis, tongue, tonsil and esophagus.

• DISEASE: Defects in DSGl are the cause of keratosis palmoplantaris striata I (PPKSl) [MIM: 148700]; also known as striate palmoplantar keratoderma I (SPPKl). PPKSl is an autosomal dominant disease characterized by thickening of the skin on the palms and soles, and longitudinal hyperkeratotic lesions on the palms, running the length of each finger.

Protein ID data

SEPARATION METHOD: 2DE

EXPECTED MOLECULAR WEIGHT/PI: 1 13716 Da / 4.90

OBSERVED MOLECULAR WEIGHT/PI: 59KD/5.8 NOTE: It could be processed the product.

2. Sequence

MDWSFFRWAVLFIFLVWEVNSEFRIQVRDYNTKNGTIKWHSIRRQKREWIKFAAACRE GEDNSKRNPIAKIHSDCAANQQVTYRISGVGIDQPPYGIFVINQKTGEINITSIVDREVT PFFIIYCRALNSMGQDLERPLξLRVRVLDINDNPPVFSMATFAGQIEENSNANTLVMIL N ATDADEPNNLNSKIAFK11RQEPSDSPMF11NRNTGEIRTMNNFLDREQYGQYALAVRGS DRDGGADGMSAECECNIKILDVNDNIPYMEQSSYTIEIQENTLNSNLLEIRVIDLDEEFS ANWMAVIFFISGNEGNWFEIEMNERTNVGILKWKPLDYEAMQSLQLSIGVRNKAEFHHS IMSQYKLKASAISVTVLNVIEGPVFRPGSKTYWTGNMGSNDKVGDFVATDLDTGRPSTT VRYVMGNNPADLLAVDSRTGKLTLKNKVTKEQYNMLGG _KY Q _G TI _LS IDDNLQRTCTGTIN INIQSFGNDDRTNTEPNTKITTNTGRQESTSSTNYDTSTTSTDSSQVYSSEPGNGAKDLL SDNVHFGPAGIGLLIMGFLVLGLVPFLMICCDCGGAPRSAAGFEPVPECSDGAIHSWAVE GPQPEPRDITTVIPQIPPDNANIIECIDNSGVYTNEYGGREMQDLGGGERMTGFELTEGV KTSGMPEICQEYSGTLRRNSMRECREGGLNMNFMESYFCQKAYAYADEDEGRPSNDCLLI YDIEGVGSPAGSVGCCSFIGEDLDDSFLDTLGPKFKKLADISLGKESYPDLDPSWPPQST EPVCLPQETEPWSGHPPISPHFGTTTVISESTYPSGPGVLHP _KP ILDPLGYGNVTVTES YTTSDTLKPSVHVHDNRPASNVWTERWGPISGADLHG _M LEMP _D LRDGSNVIVTERVIA PSSSLPTSLTIHHPRESSNVWTERVIQPTSGMIGSLSMHPELANAHNVIVTERWSGAG VTGISGTTGISGGIGSSGLVGTSMGAGSGALSGAGISGGG _IGL S _SL GGTASIGHMRSSSD HHFNQTIGSASPSTARSRITKYSTVQYSK (SEQ ID NO: 16)

3. Summary This protein is part of the desmosome cell junctions in many cell types including the heart. The protein is the antigen for Pemphigus foliaceus is an autoimmune skin disease. It

binds to plakophilin I ₁ plakophilin 2, desmoplakin, desmoglein 1, desmoglein 4, plakoglobin and corneodesmosin, all of which maybe potential biomarkers in myocardial ischemia. It has not been studied in the context of myocardial ischemia or events leading up to MI.

Q. Caspase-14

Name: Caspasc-14

IPI ID: . _. IPI00013885

UniProtKB/Swiss-Prot: P31944

Length: 242 AA [this is the length of the unprocessed precursor] propeptides 1-? AA sub-unit 1=?- 146 AA, sub-unit 2=147-242 AA Molecular Weight: 27680 Da [this is the MW of the unprocessed precursor]

1. Basic information from UniProtKB/Swiss-Prot entry P31944

• FUNCTION: May be involved in the death receptor and granzyme B apoptotic pathways. May function as a downstream signal transducer of cell death.

• SUBUNIT: May dimerize with large prodomain caspases.

• SUBCELLULAR LOCATION: Cytoplasm (By similarity).

Protein ID data

Separation method: 2DE

Expected molecular weight/pi : 27680 Da/5.44 (pro-caspase- 14=242 AA)

Observed molecular weight/pi: 68000 Da/6.8,

Note: There is difference in observed and expected MW, multiple proteins complex?

2. Sequence

MSNPRSLEEE KYDMSGARLA LILCVTKARE GSEEDLDALE HMFRQLRFES TMKRDPTAEQ FQEELEKFQQ AIDSREDPVS CAFWLMAHG RξGFLKGEDG EMVKLENLFE ALNNKNCQAL

RAKPKVYIIQ ACRGEQRDPG ETVGGDEIVM VIKDSPQTIP TYTDALHVYS TVEGYIAYRH

DQKGSCFIQT LVDVFTKRKG HILELLTEVT RRMAEAELVQ EGKARKTNPE IQSTLRKRLY LQ (SEQ ID NO: 17)

3. Summary

Caspl4 may play a role in ontogenesis and skin physiology. CASP 14 cDNA and determined that CASP 14 contains 7 exons encoding a 242-amino acid protein, 2 CASP 14 transcripts (CASP 14a and CASP 14b) differ in the C terminus while an alternative splice acceptor site within intron 5 results in a 74-nucleotide insertion in CASP 14b. CASP 14b lacks homology with the caspase consensus sequence. CASP 14 has been found in epidermis, hair follicles, the sebaceous gland. NO treatment of neonatal mouse cardiomyocytes in culture causes increase in caspase 14. There is also increase in this protein in canine brain during cardiac arrest and resuscitation. This protein has not been studied in the context of myocardial ischemia or events leading up to MI.

R. Hornerin

Name: HORNERIN

IPI ID: IPI00398625

UniProtKB/Swiss-Prot ID: Q86YZ3 Length: 2850 aa, molecular weight: 282390 D-

1. Basic information from UniProtKB/Swiss-Prot entry:

FUNCTION May play a role in cornification.

SUBCELLULAR Cytoplasmic granule (By similarity). Note=Found in keratohyalin granules

LOCATION in the granular cells of the epidermis (By similarity).

2. Sequence:

MPKLLQGVITVIDVFYQYATQHGEYDTLNKAELKELLENEFHQILKNPNDPDTVDII LQS

LDRDHNKKVDFTEYLLMIFKLVQARNKIIGKDYCQVSGSKLRDDTHQHQEEQEETEK E

ENKRQESSFSHSSWSAGENDSYSRNVRGSLKPGTESISRRLSFQRDFSGQHNSYSGQ SSS

YGEQNSDSHQSSGRGQCGSGSGQSPNYGQHGSGSGQSSSNDTHGSGSGQSSGFSQII KSS

SGQSSGYSQHGSGSGHSSGYGQHGSRSGQSSRGERHRSSSGSSSSYGQHGSGSRQSL GH

GRQGSGSRQSPSHVRHGSGSGHSSSHGQHGSGSSYSYSRGHYESGSGQTSGFGQHES GS

GQSSGYSKHGSGSGHSSSQGQHGSTSGQASSSGQHGSSSRQSSSYGQHESASRHSSG RG

QHSSGSGQSPGHGQRGSGSGQSPSSGQHGTGFGRSSSSGPYVSGSGYSSGFGHHESS SEH

SSGYTQHGSGSGHSSGHGQHGSRSGQSSRGERQGSSAGSSSSYGQHGSGSRQSLGHS RH

GSGSGQSPSPSRGRHESGSRQSSSYGPHGYGSGRSSSRGPYESGSGHSSGLGHQESR SGQ

SSGYGQHGSSSGHSSTHGQHGSTSGQSSSCGQHGATSGQSSSHGQHGSGSSQSSRYG QQ

GSGSGQSPSRGRHGSDFGHSSSYGQHGSGSGWSSSNGPHGSVSGQSSGFGHKSGSGQ SS

GYSQHGSGSSHSSGYRKHGSRSGQSSRSEQHGSSSGLSSSYGQHGSGSHQSSGHGRQ GS

GSGHSPSRVRHGSSSGHSSSHGQHGSGTSCSSSCGHYESGSGQASGFGQHESGSGQG YS

QHGSASGHFSSQGRHGSTSGQSSSSGQHDSSSGQSSSYGQHESASHHASGRGRHGSG SG

QSPGHGQRGSGSGQSPSYGRHGSGSGRSSSSGRHGSGSGQSSGFGHKSSSGQSSGYT QH

GSGSGHSSSYEQHGSRSGQSSRSEQHGSSSGSSSSYGQHGSGSRQSLGHGQHGSGSG QS

PSPSRGRHGSGSGQSSSYGPYRSGSGWSSSRGPYESGSGHSSGLGHRESRSGQSSGY GQ

HGSSSGHSSTHGQHGSTSGQSSSCGQHGASSGQSSSHGQHGSGSSQSSGYGRQGSGS GQ

SPGHGQRGSGSRQSPSYGRHGSGSGRSSSSGQHGSGLGESSGFGHHESSSGQSSSYS QHG

SGSGHSSGYGQHGSRSGQSSRGERHGSSSGSSSHYGQHGSGSRQSSGHGRQGSGSGH SP

SRGRHGSGLGHSSSHGQHGSGSGRSSSRGPYESRSGHSSVFGQHESGSGHSSAYSQH GS

GSGHFCSQGQHGSTSGQSSTFDQEGSSTGQSSSYGHRGSGSSQSSGYGRHGAGSGQS PS

RGRHGSGSGHSSSΫGQHGSGSGWSSSSGRHGSGSGQSSGFGHHESSSWQSSGCTQH GS

GSGIISSSYEQHGSRSGQSSRGERHGSSSGSSSSYGQHGSGSRQSLGHGQHGSGSGQ SPSP

SRGRHGSGSGQSSSYSPYGSGSGWSSSRGPYESGSSHSSGLGHRESRSGQSSGYGQH GSS

SGHSSTHGQHGSTSGQSSSCGQHGASSGQSSSHGQHGSGSSQSSGYGRQGSGSGQSP GH

GQRGSGSRQSPSYGRHGSGSGRSSSSGQHGSGLGESSGFGHHESSSGQSSSYSQHGS GSG

HSSGYGQHGSRSGQSSRGERHGSSSRSSSRYGQHGSGSRQSSGHGRQGSGSGQSPSR GR

HGSGLGHSSSHGQHGSGSGRSSSRGPYESRSGHSSVFGQHESGSGHSSAYSQHGSGSGH ^~

FCSQGQHGSTSGQSSTFDQEGSSTGQSSSHGQHGSGSSQSSSYGQQGSGSGQSPSRG RH

GSGSGHSSSYGQHGSGSGWSSSSGRHGSGSGQSSGFGHHESSSWQSSGYTQI-IGSG SGHS

SSYEQHGSRSGQSSRGEQHGSSSGSSSSYGQHGSGSRQSLGHGQHGSGSGQSPSPSR GR

HGSGSGQSSSYGPYGSGSGWSSSRGPYESGSGHSSGLGHRESRSGQSSGYGQHGSSS GH

SSTIIGQHGSASGQSSSCGQHGASSGQSSSHGQHGSGSSQSSGYGRQGSGSGQSPGH GQ

RGSGSRQSPSYGRHGSGSGRSSSSGQHGPGLGESSGFGHHESSSGQSSSYSQHGSGS GHS

SGYGQHGSRSGQSSRGERHGSSSGSSSRYGQHGSGSRQSSGHGRQGSGSGHSPSRGR HG

SGSGHSSSHGQHGSGSGRSSSRGPYESRSGHSSVFGQHESGSGHSSAYSQHGSGSGH FCS

QGQHGSTSGQSSTFDQEGSSTGQSSSHGQHGSGSSQSSSYGQQGSGSGQSPSRGRHG SG

SGHSSSYGQHGSGSGWSSSSGRHGSGSGQSSGFGHHESSSWQSSGYTQHGSGSGHSS SY

EQHGSRSGQSSRGERHGSSSGSSSSYGQHGSGSRQSLGHGQHGSGSGQSPSPSRGRH GS

GSGQSSSYSPYGSGSGWSSSRGPYESGSGHSSGLGHRESRSGQSSGYGQHGSSSGHS STH

GQHGSTSGQSSSCGQHGASSGQSSSHGQHγTSπSSQSSGYGRQGSGSGQSPGHGQ RGSG

SRQSPSYGRHGSGSGRSSSSGQHGSGLGESSGFGHHESSSGQSSSYSQHGSGSGHSS GYG

QHGSRSGQSSRGERHGSSSGSSSHYGQHGSGSRQSSGHGRQGSGSGQSPSRGRHGSGLG HSSSHGQHGSGSGRSSSRGPYESRLGHSSVFGQHESGSGHSSAYSQHGSGSGHFCSQGQ HGSTSGQSSTFDQEGSSTGQSSSYGHRGSGSSQSSGYGRHGAGSGQSLSHGRHGSGSGQ SSSYGQHGSGSGQSSGYSQHGSGSGQDGYSYCKGGSNHDGGS _. SGSYFLSFPSSTSPYEY VQEQRCYFYQ (SEQ ID NO: 18)

Little is known about this protein. It has not been studied in the context of myocardial ischemia or events leading up to MI.

SLKiniπogen

Name: ISOFORM LMW OF KININOGEN-I

IPI ID: IPI00215894

UniProtKB/Swiss-Prot ID: PO 1042 Length: 427 aa, molecular weight: 47883 Da (of Precursor) t. Basic information from UniProtKB/Swiss-Prot entry:

(1) Kininogens are inhibitors of thiol proteases; (2) HMW-kininogen plays an important role in blood coagulation by helping to position optimally prekallikrein and factor XI next to factor XII; (3) HMW-kininogen inhibits the thrombin- and plasmin- induced aggregation of thrombocytes; (4) the active peptide bradykinin that is released from HMW-kininogen shows a variety of physiological effects: (4A) influence in smooth muscle contraction, (4B) induction of hypotension, (4C) FUNCTION natriurcsis and diuresis, (4D) decrease in blood glucose level, (4E) it is a mediator of inflammation and causes (4El) increase in vascular permeability, (4E2) stimulation of nociceptors (4E3) release of other mediators of inflammation (e.g. prostaglandins), (4F) it has a cardioprotective effect (directly via bradykinin action, indirectly via endothelium-derived relaxing factor action); (5) LMW-kininogen inhibits the aggregation of thrombocytes; (6) LMW- kininogen is in contrast to MMW-kininogen not involved in blood clotting.

SUBCELLULAR Secreted, extracellular space. LOCATION

2. Sequence:

MKLITILFLCSRLLLSLTQESQSEEIDCNDKDLFKAVDAALKKYNSQNQSNNQFVLY RIT EATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTK FSVATQTCQITPAEGPVVTAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFM LNEVKRAQRQVVAGLNFRITYSIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDI QLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNλT FYF KIDNVKKλRVQVVAGKKYFIDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVYV VPWEKKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGEIKEETTSHLRSCEYKGRPPKAG A EPASEREVS (SEQ ID NO: 19)

3. Alternative name(s): High molecular weight kininogen, Short name=HMWK; Williams-Fitzgerald- Flaujeae factor; Fitzgerald factor; Alpha-2-thiol proteinase inhibitor

This protein has not been studied in the context of myocardial ischemia or events leading up to MI.

Example III. Further studies to identify cardiac biomarkers, using as a cohort of patients, a valve replacement cardioplegia human model (cohort II)

A. Overview of the studies

The 21 patients in this cohort all underwent aortic valve replacement surgery (See Figures 6 and 7). Table 10 provides cohort information of this model.

Table 10: patient sampling

Tl pre-op sample (before incision).

T2 immediately before the heart gets stopped (prior to CPB).

T6 5 min after the heart went off CPB and was beating on its own again.

T7 30 min after bypass

T8 60 min after bypass

T9 120 min after bypass

There were 6 plasma samples taken. Note that only 19 (out of 21) patients have samples at T9. The sample were normalized to total protein concentration for both targeted and de novo discovery. In the targeted analysis, 15 biomarkers were determined for.each sample, specifically, CRP ₁ GM-CSF, IFNγ, ILlO ₅ !L12p70, ILl β, IL-2, IL-6, IL-8, NT proBNP, SAA, TNFα, cTnl, sICAM, sVCλM. All time points were analyzed. These analyses were done using the MESOSCALE multiplex assay in triplicate. Figure 7 shows the box blot for cTnl measurement for all patients. For de novo discovery, only T2 and T6 were analyzed.

B. Methods

1. High abundant protein depletion

IgY depletion of the top 12 abundant proteins of samples from each individual sample.

2. Intact protein separation by hydrophobicity

1 DLC analysis was carried out for cohort II (20 patients with two time points). All IDLC analyses were done in duplicate using the optimized gradient developed to eliminate the interference of the unknown contaminates that eluted at the beginning and end of the run. Optimization was required as the contaminants were not MS compatible and co-eluted with proteins found to be interesting in cohort I. The duplicate run for each patient time point were collected into a single plate and stored at -8O ⁰ C until analyzed. A total of 80 @ IDLC runs were carried out (20 x 2 x 2. = 80 (2 time points, 20 patients, in duplicate)). The fractions obtained for each IDLC run were pooled into 16 fractions, neutralized and dried down, prior to resolublization in buffer compatible for tryptic digestion.

3. MS analysis

MS analysis for each digested fraction was carried using the LTQ Orbitrap LC MSMS instrument. Each fraction was run in duplicate. A total 1216 MS runs were carried out (19 x 2 x 16 x 2 = 1216 (19 patients, 16 fractions per time point, two time points, in duplicate). For LC- MS/MS experiments on the LTQ-Orbitrap (ThermoFinnigan, San Jose, CA), peptides were dissolved in 6 μl resuspension buffer (4% acetonitrile in water with 0.1% formic acid). Samples (3 μl) were loaded onto a 75um x 10 cm BioBasic Cl 8 column (New Objective, Woburn, MA). Peptides were eluted into an LTQ-Orbitrap (ThermoFinnigan, San Jose, CA) using an Agilent 1200 nano-LC system (Agilent, Santa Clara, CA). The HPLC gradient was 5% to 60% B (90% acetonitrile/water in 0.1% Formic acid) over 30 or 60 min depended on sample complexity. The mass spectrometer was operated in data-dependent mode in which every FT-MS scan (survey 350-2000 Da) was followed by MS/MS scans of the 5 most abundant ions.

Mass spectrometry data were analyzed, and data reanalysis was carried out, as described in Example 1D2 above.

C. Results

1. Optimization of IDLC

In order for cohort II to be analyzed, optimization of the IDLC gradient was required to resolve a contaminating peak eluting early on the chromatogram. The contaminating peak overlaid a region in which we found several potential biomarkers and resulted in suppression of the peptides of interest.

2.Optimization and testing of reproducibility of MS analysis

In table 1 1, the MS reproducibility of several fractions is shown.

Ul

Ul Ul

This space jntefifϊonally left blank.

3. Cohort II analysis

343 non-redundant proteins were compared. Table 12 shows those proteins which were most significantly increased in T6 compared to TO for cohort II.

Table 12: Target proteins in cohort II

Table 12A: Top hits from cohort II clustered based on related protein family. Below listed the number of individuals the protein was elevated in T6 compared to T2 (increased due to induced ischemia). 20 individuals were analyzed. 1. PRDX2 Peroxiredoxin 2 - increased in 17 patients

2. Sl 00 A9 Protein S 100-λ9 - increased in 17 patients SlOO A8 - increased in 11 patients

S 100 A7- increased in 4 patients

3. Lactotransferrin increased in 14 patients 4. CAl Carbonic anhydrase 1 - increased in 17 patients

CA2 Carbonic anhydrase 2 - increased in 3 patients

5. Conserved hypothetical protein - increased in 12 patients

6. NCOR2 CTG26 alternate open reading frame (Fragment) - increased in 11 patients

7. LOC729968 Conserved hypothetical protein - increased in 11 patients 8. Conserved hypothetical protein - increased in 9 patients

9. SORLl Sortilin-related receptor - increased in 11 patients

10. COLlAl Collagen alpha- 1(1) chain precursor - increased in 11 patients COL 1A2 130 kDa protein - increased in 10 patients

11. CPB2 Isoform 1 of Carboxypeptidase B2 precursor - increased in 10 patients Carboxypeptidase subunit 2 - increased in 8 patients

12. HBB Hemoglobin subunit beta - increased in 12 patients HBA Hemoglobin subunit alpha - increased in 4patients

13. Lactotransferrin - increased in 12 patients

14. LRPl Prolow-density lipoprotein receptor-related protein 1 precursor - increased in 9 patients

LRP2 Low-density lipoprotein receptor-related protein 2 precursor - increased in 9 patients

Together a total of 13 unique patients

15. CAT Catalase - increased in 11 patients 16. STX3 Isoform. A of Syntaxin-3 - increased in 9 patients

17. ECMl Extracellular matrix protein 1 - increased in 10 patients

18. SERPINAlO Protein Z-dependent protease inhibitor precursor - increased in 8 patients

19. PTPRK Isoform 1 of Receptor-type tyrosine-protein phosphatase kappa precursor increased in 8 patients 20. ATRN Isoform 2 of Attractin precursor - increased in 8 patients

21. PTPRK Protein tyrosine phosphatase, receptor type, K increased in 6 patients

Table 12B: Others of interest due to biology (there are low abundant proteins and may not be observed in higher number of patients due to detection issues)

22. MSTl Hepatocyte growth factor-like protein precursor - increased in 7 patients HGFAC Hepatocyte growth factor activator precursor - increased in 4 patients

23. IGFBP6 Insulin-like growth factor-binding protein 6 precursor - increased in 5 patients IGFBP5 Insulin-like growth factor-binding protein 5 precursor - increased in 1 patients IGFBP2 Insulin-like growth factor-binding protein 2 precursor - increased in 7 patients

(same ones as 7)

IGFBP7 Insulin-like growth factor-binding protein 7 precursor - increased in 7 patients

(same ones as 2)

IGFALS Insulin-like growth factor-binding protein complex acid labile chain precursor - increased in 3 patients

Total for group 8 patients

24. SERPINFl Pigment epithelium-derived factor precursor - increased in 4 patients

25. GPX3 Glutathione peroxidase 3 precursor - increased in 4 patients

26. CD 14 Monocyte differentiation antigen CDl 4 precursor - increased in 4 patients

27. Note there are interesting proteins seen in 2-3 patients which may still be important alternations which are not seen in more patients due to their low abundance. These should be discussed.

A summary of some of the properties of these proteins is presented in Example IV.

4. Cohort II and I comparison

Comparison of the top candidate proteins between cohort I and II are shown in Table 13. Table 13 Tier One

Protein IPI number Cohort Cohort Il FUNCTION Is protein

I secreted?

Lumican . IPI00020986 Top Found May be involved in cell Yes response to injury

Extracellular matrix IPI00645849 Top Found Involved in extracellular Yes protein matrix compostition

Carboxypeptidase IPI00010295 Mid Found Protease involved in Yes

N catalytic chain regulation of vasoactive and inflammatory peptides angiogenin IPI00008554 Top no May be involved in Yes angiogeneisis ^• semenogelin IPI00414684 Top no Forms gel matrix around Yes sperm, unknown role in other cells

Lung PLNECA-1 IPI00291410 Top no May play a role in innate Yes immunity

Perioxiredoxln 2 IPI00027350 No Elevated Involved in redox regulation of No in 17, Top the cell.

S100 A9 IPI00027462 No Elevated Expressed by macrophages in No in 17, Top acutely inflamed tissues and in chronic inflammations.

S100 A8 IPI00007047 No Elevated Expressed by macrophages in unknown in 11 , Top chronic inflammations. Also expressed in epithelial cells constitutively or induced during dermatoses.

S100 A7 IPI00219806 No Elevated unknown Secreted in 4, Lower

Sortilin-related IPI00022608 No Elevated Likely to be a multifunctional No, is a plasma receptor in 11, Mid endocyf c receptor, which membrane protein implicates it in the uptake of lipoproteins and of proteases.

Catalase IPI00465436 No Elevated Serves to protect cells from the No in 11 , Mid toxic effects of hydrogen peroxide.

Low density IPI00020557 No Elevated Endocytic receptor involved in No lipoprotein receptor in 9, Mid endocytosis and in phagocytosis related protein 1 of apoptotic cells. Low density IPI00024292 No Elevated Acts together with cubϊlin to No lipoprotein receptor in 9, Top mediate HDL endocytosis (By related protein 2 similarity). Syntaxin-3 IPI00395768 No Evelated Potentially involved in docking of No In 9, Mid synaptic vesicles at presynaptic active zones.

Tier two

Hepatocyte growth IPI00292218 + Unknown Unknown factor like protein Hepatocyte growth IPI00029193 + Activates hepatocyte growth Secreted. factor activator factor (HGF). Insulin like growth IPI00029235 + GF-binding proteins prolong the Secreted. factor protein 6 half-life of the IGFs. Pigment epithelium- IPI00006114 + Neurotrophic protein; induces Secreted. derived factor extensive neuronal differentiation in retinoblastoma cells. Potent inhibitor of aπgiogenesis.

Glutathione IPI00026199 + Protects cells and enzymes from Secreted . peroxidase 3 oxidative damage. Monocyte IPI00029260 + Involved in the innate immune No differentiation response to bacterial antigen CD14 lipopolysaccharide (LPS). Lactotransferrin, IPI00789477 + unknown unknown cDNA FLJ58679, highly similar to Lactotransferrin

Attractin IPI00162735 Involved in the initial immune cell Secreted clustering during inflammatory response.

Conserved IPI00883661 Unknown Unknown hypothetical protein NC0R2 CTG26 IPI00006659 unknown unknown alternate open reading frame LOC729968 IPI00884334 unknown unknown Conserved hypothetical protein Protein Z- IPI00007199 Inhibits factor Xa activity. Secreted, dependent protease inhibitor Conserved IPI00847894 unknown unknown hypothetical protein lsoform 1 of IPI00015756 Regulation of processes No ₁ but present Receptor-type involving cell contact and on plasma tyrosine-protein adhesion such as growth control, membrane phosphatase kappa ^♦iinri or invasion, and metastasis.

Protein tyrosine IPI00552690 Receptor Unknown, but phosphatase, present on receptor type, K plasma membrane

Sodium channel IPI00217376 Elevated Part of sodium channel No, but present subunit beta4 in 7 on plasma people membrane

Alpha2-HS- IPI00022431 + Elevated See previous write up for See previous glycoprotein in 4 cohort I write up for people cohort I

Galectin 7 IPI00219221 + See previous write up for See previous cohort I write up for cohort I

Hornerin IPI00398625 + Elevated See previous write up for See previous and seen cohort I write up for only in cohort I one person

Proteoglycan 4 IPI00655676 + Elevated See previous write up for See previous (isoforms A and D) in 4 cohort I write up for people cohort I

Proflaggrin IPI00654788 + See previous write up for See previous (Filaggrin) cohort I write up for cohort I

Vitamin D binding IPI00555812 + See previous write up for See previous protein cohort I write up for cohort I

C4b binding IPI00021727 + See previous write up for See previous proteins cohort I write up for cohort I

Thyroxine binding IPI00292946 + See previous write up for See previous globulin cohort I write up for cohort I

Alpha 2 IPI00166729 + Elevated See previous write up for See previous glycoprotein 1 , zinc in 3 cohort I write up for people cohort I

Caspase 14 + See previous write up for See previous cohort I write up for cohort I

Desmogelin See previous write up for See previous cohort I write up for cohort I

Kininogen -1 IPI00215894 + See previous write up for See previous cohort I write up for cohort I

Some additional proteins were found to be elevated in a subset of the patients in cohort II that exhibit ischemia. See Table 14 below for details.

# individal with ischemic

Protein induced

Table 14 accession increase

Protein name numbers (max. 20)

PRDX2 Peroxiredoxiπ-2 IPI00027350 17

S100A9 Protein S100-A9 IPI00027462 17

LTF Similar to Lactotransfem ^' n IPI00789477 14

Conserved hypothetical protein IPI00883661 12

HBB Hemoglobin subunit beta IPI00654755 12

NCOR2 CTG26 alternate open reading frame IPI00006659 11

S100A8 Protein S100-A8 IPI00007047 11

SORL1 Sortilin-related receptor IPI00022608 11

CA1 Carbonic anhydrase 1 IPI00215983 11

COL1A1 Collagen alpha-1 (I) chain IPI00297646 11

CAT Catalase IPI00465436 11

ALB lsoform 1 of Serum albumin IPI00745872 11

LOC729968 Conserved hypothetical protein IPI00884334 11

CFI Complement factor I IPI00291867 10

CPB2 lsoform 1 of Carboxypeptldase B2 IPI00329775 10

Ig heavy chain V-Il region OU IPI00382534 10

Ig kappa chain V-I region Ka IPI00387095 10

COL1A2 13O kDa protein IPI00873137 10

ECM 1 Extracellular matrix protein 1 IPI00645849 10

LRP1 Prolow-density lipoprotein receptor-related protein 1 IPI00020557 9

LRP2 Low-density lipoprotein receptor-related protein 2 IPI00024292 9

C7 Complement component C7 IPI00296608 9

STX3 lsoform A of Syntaxiπ-3 IPI00395768 9

SERPINA1 lsoform 1 of Alpha-1 -antitrypsin IPI00553177 9

LOC440786 Ig kappa chain V-Il region

TEW IPI00736885 9

Conserved hypothetical protein IPI00847894 9

SERPIN A10 Protein Z-dependent protease inhibitor IPI00007199 8

PTPRK lsoform 1 of Receptor-type tyrosine-protein phosphatase kappa IPI00015756 8

AMBP AMBP protein IPI00022426 8

TF Serotransferrin IPI00022463 8

Cδ Complement C5 IPI00032291 8

ATRN lsoform 2 of Attractin IPI00162735 8

C1QB complement component 1, q subcomponent, B chain IPI00477992 8

CPN2 Carboxypeptldase N subunit 2 IPI00479116 8

SERPINA5 Plasma serine protease inhibitor IPI00007221 7

LUM Lumican IPI00020986 7

APOB Apolipoprotein B-100 IPI00022229 7

C1QC Complement C1q subcomponent subunit C IPI00022394 7

SHBG lsoform 1 of Sex> hormone-binding globulin IPI00023019 7

SCN4B lsoform 1 of Sodium channel subunit beta-4 IPI00217376 7 MST1 Hepatocyte growth factor-like protein IPI00292218 7 MDFI 19 kDa protein IPI00385435 7

QSOX1 lsoform 2 of Sulfhydryl oxidase 1 IPI00465016 7

FETUB GUGU beta form IPI00552199 7

SEPP1 selenoprotein P isoform 2 IPI00847381 7

HBA2;HBA1 Alpha 2 globin variant (Fragment) IPI00853068 7

CPN1 Carboxypeptidase N catalytic chain IPI00010295 6

AFM Afamin IPI00019943 6

SOD1 Superoxide dismutase IPI00218733 6

VTN Vitronectin IPI00298971 6

SERPINA4 Kallistatin IPI00328609 6

SERPINA4 Kallistatin IPI00328609 6

PTPRK Protein tyrosine phosphatase, receptor type, K IPI00552690 6

SERPINA3 Isoform 1 of Alpha-1-antichymotrypsin IPI00847635 6

OU domain class 5 transcription factor 1 (Fragment) IPI00868800 6

ORM1 orosomucoid 1 IPI00884926 6

F12 Coagulation factor XII IPI00019581 5

APOA1 Apolipoprotein A-I IPI00021841 5

IGFBP6 Insulin-like growth factor-binding protein 6 IPI00029235 5

FN 1 Isoform 3 of Fibronectin IPI00339223 5 g heavy chain V-III region CAM IPI00382482 5

LOC388720 similar to ubiquitin and ribosomal protein S27a IPI00397808 5

CLU clusterin isoform 1 IPI00400826 5

BTD Uncharacterized protein BTD (Fragment) IPI00744685 5

PROS1 80 kDa protein IPI00873445 5

SERPINF1 Pigment epithelium-derived factor IPI00006114 4

C8G Complement component C8 gamma chain IPI00011261 4

0RM1 Alpha-1-acid glycoprotein 1 IPI00022429 4

AHSG Alpha-2-HS-glycoprotein IPI00022431 4

GGH Gamma-glutamyl hydrolase IPI00023728 4

EFNB1 Ephrin-B1 IPI00024307 4

GPX3 Glutathione peroxidase 3 IPI00026199 4

HGFAC Hepatocyte growth factor activator IPI00029193 4

CD14 Monocyte differentiation antigen CD14 IPI00029260 4

FGA Isoform 2 of Fibrinogen alpha chain IPI00029717 4

LRP1 B Similar to Candidate tumor suppressor protein IPI00032063 4

S100A7 Protein S100-A7 IPI00219806 4

C8B Complement component C8 beta chain IPI00294395 4

DMXL1 DmX-like protein 1 IPI00294728 4

ARSB Arylsulfatase B IPI00306576 4

LRP8 lsofbrm 3 of Low-density lipoprotein receptor-related protein 8 IPI00384247 . 4

HBA2;HBA1 Hemoglobin subunit alpha IPI00410714 4

ASPN ASPN protein IPI00418431 4

A2M Alpha-2-macroglobulin IPI00478003 4

CDH3 lsofomn 2 of Cadherin-3 IPI00645614 4

KLKB1 Plasma kallikrein IPI00654888 4

SERPINA1 lsoform 2 of Alpha-1 -antitrypsin IPI00790784 4

ICAM2 28 kDa protein IPI00793958 4

C1RL cDNA FLJ14022 fis, clone HEMBA1003538, weakly similar to COMPLEMENT C1 R COMPONENT IPI00795055 4

B2M B2M protein IPI00796379 4

APOA1 Apolipoprotein A1 IPI00853525 4

Transthyretin IPI00855916 4

ITIH3 Uπcharacterized protein ITIH3 IPI00873416 4

MB 16 kDa protein IPI00878623 4

SERPINF2 Alpha-2-antiplasmin IPI00879231 4

COL5A2 Collagen alpha-2(V) chain IPI00844306 4

JUP Junction plakoglobin IPI00554711 4

PRG4 lsoform D of Proteoglycan-4 IPI00655676 4

GPR37 Probable G-protein coupled receptor 37 IPI00006166 3

F13B Coagulation factor XIII B chain IPI00007240 3

CLEC3B Tetranectin IPI00009028 3

C6 Complement component 6 IPI00009920 3

C8A Complement component C8 alpha chain IPI00011252 3

DSP lsoform DPI of Desmoplakiπ IPI00013933 3

COPS2 lsoform 2 of COP9 signalosome complex subunit 2 IPI00018813 3

F2 Prothrombin (Fragment) IPI00019568 3

IGFALS Insulin-like growth factor-binding protein complex acid labile chain IPI00020996 3

ACTG1 Actin, cytoplasmic 2 IPI00021440 3

FGA lsoform 1 of Fibrinogen alpha chain IPI00021885 3

RBP4 Plasma retinol-binding protein IPI00022420 3

RBP4 Retinol binding protein 4, plasma IPI00022420 3

HPX Hemopexin IPI00022488 3

NPR1 Atrial natriuretic peptide receptor A IPI00027200 3

SEPP1 Selenoprotein P IPI00029061 3

ZAK lsoform 2 of Mitog en-activated protein kinase kinase kinase

MLT IPI00029643 3

CST3 Cystatin-C IPI00032293 3

AZGP1 alpha-2-glycoprotein 1, zinc IPI00166729 3

CA2 Carbonic anhydrase 2 IPI00218414 3

SELL L-selectin IPI00218795 3

FGG lsoform Gamma-A of Fibrinogen gamma chain IPI00219713 3

IGSF5 Immunoglobulin superfamily member 5 IPI00245940 3

LYVE1 Lymphatic vessel endothelial hyaluronic acid receptor 1 IPI00290856 3

SERPING1 Plasma protease C1 inhibitor IPI00291866 3

C17orf13,ACYP1,C1R Complement C1r subcomponent IPI00296165 3

F9 Coagulation factor IX IPI00296176 3

FGB Fibrinogen beta chain IPI00298497 3

LILRB2 leukocyte immuπoglobulin-like receptor, subfamily B, member 2 isoform 1 IPI00303952 3

IGHG1 Putative uncharacteπzed protein DKFZp686N02209 IPI00384938 3 cDNA FLJ43303 fis, clone NOVAR2000136, moderately similar to Calsequestrin, skeletal muscle isoform I Pl 00445889 3

IGHM IGHM protein IPI00472610 3

PTGDS Prostaglandin D2 synthase 21kDa IPI00513767 3

GC Vitamin D-binding protein IPI00555812 3

HP Haptoglobin IPI00641737 3

LCN2 Lipocalin 2, Neutrophil gelatiπase-associated lipocalin IPI00643623 3

ITIH2 Inter-alpha (Globulin) inhibitor H2 IPI00645038 3

FETUB GUGU beta form, Fetuιn-B IPI00743766 3

HABP2 Hyaluronan-binding protein 2 IPI00746623 3

C1S Uncharacterized protein C1S IPI00749179 3

LRP1B Low-density lipoprotein receptor-related protein 1B IPI00877809 3

SERPIND1 Heparin cofactor 2 IPI00879573 3

APOA4 Apolipoprotein A-IV I Pl 00304273 3

CDH22 Cadherin-22 IPI00000436 2

TAF9 Transcription initiation factor TFIID subunit 9 IPI00002993 2

MBL2 Mannose-binding protein C IPI00004373 2

CRISP3 cDNA FLJ75207 IPI00004798 2

EFNA4 Isoform 1 of Ephπn-A4 IPI00005125 2

COMT Isoform Membrane-bound of Catechol O- methyltransferase IPI00011284 2

LRRC4C Netrιn-G1 ligand IPI00014223 2 _C M _{I C} M _C M _CS

IGFBP7 Insulin-like growth factor-binding protein 7 IPI00016915 2

C1S Complement C1s subcomponent IPI00017696 2

GGT1 Isoform 1 of Gamma-glutamyltranspeptidase 1 IPI00018901

PLG Plasminogen IPI00019580 2

0RM2 Alpha-1-acιd glycoprotein 2 IPI00020091 2

PLXNA3 Plexιn-A3 IPI00020884 2

C4BPA C4b-bιnding protein alpha chain IPI00021727 2

SERPINB3 Serpin B3 IPI00022204

BAM Brain-spectfic angiogenesis inhibitor 1 IPI00022333 2

HRG Histidine-rich glycoprotein IPI00022371 2

APCS Serum amyloid P-component IPI00022391

C1QA Complement C1q subcomponent subunit A IPI00022392

C9 Complement component C9 IPI00022395

NRGN Neurogranin IPI00022640 2

CDH1 Epithelial cadherin IPI00025861 2

SOD3 Extracellular superoxide dismutase [Cu-Zn] IPI00027827 2

KNG1 Isoform HMW of Kιnιnogen-1 IPI00032328 2

PTH2 Tuberoinfuπdibular peptide of 39 residues IPI00059307 2

PTPRU protein tyrosine phosphatase, receptor type, U isoform 3 IPI00107472 2

PTPRF Receptor-type tyrosine-protein phosphatase F IPI00107831 2

UBA52 ubiquitin and ribosomal protein L40 , UBB;RPS27A;UBC ubiquitin and ribosomal protein S27a IPI00179330 2

UBB;RPS27A;UBC ubiquitin and ribosomal protein S27a IPI00179330 2

TTN lsoform 7 of Titin IPI00179357 2

CLCN6 lsoform A of Chloride channel protein 6 IPI00180121 2

HIST1H1C Histone H1.2 IPI00217465 2

MB Myoglobin IPI00217493 2

HRC Sarcoplasmic reticulum histidine-rich calcium-binding protein IPI00219226 2

SP140 lsoform LYSpIOO-A of Nuclear body protein SP140 IPI00219535 2

PTPRO Receptor-type tyrosine-protein phosphatase O IPI00241041 2

CLU Clusterin IPI00291262 2

SLC44A2 lsoform 2 of Choline transporter-like protein 2 IPI00293074 2

ITIH4 lsoform 1 of Inter-alpha-trypsin inhibitor heavy chain H4 IPI00294193 2

IGFBP2 Insulin-like growth factor-binding protein 2 IPI00297284 2

LTF Growth-inhibiting protein 12 IPI00298860 2

LCN2 Neutrophil gelatinase-associated lipocalin IPI00299547 2

THBS4 Thrombospondin-4 IPI00328550 2

Ig heavy chain V-III region TEI IPI00382494 2

VASN Vasorin IPI00395488 2

FLG2 lfapsoriasin IPI00397801 2

SEMG1 lsoform 2 of Semenogelin-1 IPI00414684 2

IGHG1 Putative uncharacterized protein DKFZp686N02209 IPl00423466 2

HUWE1 lsoform 2 of E3 ubiquitin-proteln ligase HUWE1 IPI00445401 2

PTPRK Protein tyrosine phosphatase, receptor type, K IPI00470937 2

HP HP protein IPI00478493 2

FCGR3A Fc fragment of IgG, low affinity Ilia, receptor for IPI00640044 2

C2 Complement component 2 IPI00643506 2

CPN2 similar to Carboxypeptidase N subunit 2 IPI00738433 2

A1 BG alpha 1 B-glycoprotein IPI00745089 2

LOC732428 Uncharacterized protein ENSP00000375150 IPI00787862 2

SOD1 Uncharacterized protein SOD1 IPI00789078 2

CLEC3B Putative uncharacterized protein DKFZp686H 17246 IPI00792115 2

3 kDa protein IPI00792845 2

FCGR3B Protein IPI00795501 2

C5 Complement component 5 variant (Fragment) IPI00816741 2

PRAP1 lsoform 4 of Proline-rich acidic protein 1 IPI00855875 2

SH3BGRL 13 kDa protein IPI00872670 2

Example IV. Summary of some of the properties of proteins discussed with regard to cohort II.

A. Pigment Epithelium-Derived Factor

Name: PIGMENT EPITHELIUM-DERIVED FACTOR

IPI ID: IPI00006114

UniProtKB/Swiss-Prot ID: P36955 Length: 418 aa, molecular weight: 46342 Da

1. Basic information from UniProtKB/Swiss-Prot entry:

Neurotrophic protein; induces extensive neuronal differentiation in T _XT PY _TZ - _VX J retinoblastoma cells. Potent inhibitor of angiogenesis. As it does not undergo the

-"-^ ^ — ^^ S (stressed) to R (relaxed) conformational transition characteristic of active serpins, it exhibits no serine protease inhibitory activity.

SUBCELLULAR Secreted. Melanosome. Notc=Enriched in stage I melanosomes. LOCATION pyv. The N-terminus is blocked. Extracellular phosphorylation enhances antiangiogenic activity.

2. Sequence:

MQALVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAA VS

NFGYDLYRVRSSMSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSP DIHG

TYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDL QEI

NNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEER T

VRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTS EFIH

DIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQV EHR

AGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPR GP

(SEQ ID NO:20)

3. Alternative name(s): Serpin-Fl, EPC-I

Has been used in treatment of retinal ischemic injury. As well, increased levels are observed with retinal diseases and diabetes but none have been related to heart disease including myocardial ischemia.

B. Protein S100-A7

Name: Protein S 100-A7 IPI ID: IPI00219806 UniProtKB/Swiss-Prot ID: P31151 Length: 101 aa, molecular weight: 11471 Da

1. Basic information from UniProtKB/Swiss-Prot entry:

Subcellular location Cytoplasm. Secreted.

Subunit structure Interacts with RANBP9.

2. Sequence:

MSNTQAERSIIGMIDMFHKYTRRDDKIEKPSLLTMMKENFPNFLSACDKKGTNYLAD VF EKKDKNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPCSGGSQ (SEQ ID NO:21)

3. Alternative nanie(s): SlOO calcium-binding protein A7; Psoriasin

This protein has not been linked to myocardial ischemia or events leading up to MI.

C. Protein S100-A8

Name: Protein Sl OO A8 IPI ID: IPI00007047 UniProtKB/Swiss-Prot ID: P05109 Length: 93 aa, molecular weight: 10835 Da

1. Basic information from UniProtKB/Swiss-Prot entry:

• Function: Expressed by macrophages in chronic inflammations. Also expressed in epithelial cells constitutively or induced during dermatoses. May interact with components of the intermediate filaments in monocytes and epithelial cells.

2. Sequence:

MLTELEKALNSIIDVYHKYSLIKGNFHAVYRDDLKKLLETECPQ YIRKKGADVWFKELD INTDGAVNFQEFLILVIKMGVAAHKKSHEESHKE (SEQ ID NO:22)

3. Alternative name(s):

S 100 calcium-binding protein A8 Calgranulin-A Migration inhibitory factor-related protein 8

Short name=MRP-8

Short name=P8 Cystic fibrosis antigen

Short name=CFAG Leukocyte Ll complex light chain Calprotectin LlL subunit Urinary stone protein band A

This protein has not been linked to myocardial ischemia or events leading up to MI.

D. Protein SI 00-A9

Name: PROTKIN sioo-A9

IPI ID: IP100027462 UniProtKB/Swiss-Prot ID: P06702 Length: 1 14 aa, molecular weight: 13242 Da

1. Basic information from UniProtKB/Swiss-Prot entry:

• Function: Expressed by macrophages in acutely inflammated tissues and in chronic inflammations. Seem to be an inhibitor of protein kinases. Also expressed in epithelial cells constitutively or induced during dermatoses. May interact with components of the intermediate filaments in monocytes and epithelial cells.

• Subcellular location: cytoplasm and nucleus.

2. Sequence:

MTCKMSQLERNIETIINTFHQYSVKLGHPDTLNQGEFKELVRKX)LQNFLKKENKNE KVI EHIMEDLDTN ADKQLSFEEFIMLMARLTWASHEKMHEGDEGPGHHHKPGLGEGTP (SEQ ID NO:23)

3. Alternative name(s):

FuIl=SlOO calcium-binding protein A9;

Full=Calgranulin-B;

Full=Migration inhibitory factor-related protein 14;

Short=MRP- 14;

Shorc=P 14;

Full=Leukocyte Ll complex heavy chain;

Full=Calprotectin LlH subunit;

The mRNA levels of SlOO A9 have been shown to increase after ischemic brain injury and after stroke. The protein level was not detemined. This protein has not been linked to myocardial ischemia or events leading up to MI.

E. Protein tyrosine phosphatase, receptor type. K

Name: PROTEIN TYROSINE PHOSPHATASE, RECEP TOR TYPE, K

IPI ID: IPI00552690

UniProtKBλfrEMBL ID: Q5JY45

Length: 202 aa, molecular weight: 22792 Da

Sequence:

MSSVEKETKTQCVRIATKAλATEEPEVIPDPAKQTDRVVKIAGISAGILVFILLLL VVILIV KKRRSYYSYSYYLKLAKKRKDAMGNTRQEMTHMVNAMDRSYADQSTLHAEDPLSITF MDQHNFSPRLPNDPLVPTAVLDENHSATAESSRLLDVPRYLCEGTESPYQTGQLHPAIR VADLLQHINLMKTSDSYGFKEEYE (SEQ ID NO:24)

This protein has not been linked to myocardial ischemia or events leading up to MI.

E, Protein Z-dependent protease inhibitor

Name: Protein Z-dependent protease inhibitor

IPI ID: IPI00007199

UniProtKB/Swiss-Prot ID: Q9UK55 Length: 484 aa, molecular weight: 55114 Da (of Precursor)

1. Basic information from UniProtKB/Swiss-Prot entry:

Inhibits factor Xa activity in the presence of protein Z, calcium and r UNC I IOiN . , .. . . phospholipid.

SUBCELLULAR Secreted

LOCATION

2. Sequence:

MSRSTQELLGYHCRLQDKLQEQEGSLAAEGRHSLASAADHMKVVPSLLLSVLLAQVW

LVPGLAPSPQSPETPAPQNQTSRVVQAPKEEEEDEQEASEEKASEEEKAWLMASRQQ LA

KETSNFGFSLLRKISMRHDGNMVFSPFGMSLAMTGLMLGATGPTETQIKRGLHLQAL K

PTKPGLLPSLFKGLRETLSRNLELGLTQGSFAFIHKDFDVKETFFNLSKRYFDTECV PMN

FRNASQAKRLMNHYINKETRGKIPKLFDEINPETKLIL VDYILFKGKWLTPFDPVFTEVD

TFIILDKYKTIKVPMMYGAGKFASTFDKNFRCHVLKLPYQGNATMLVVLMEKMGDHL

ALEDYLTTDLVETWLRNMKTRNMEVFFPKFKLDQKYEMHELLRQMGTRRIFSPFADL S

ELSATGRNLQVSRVLQRTVIEVDERGTEAVAGILSEITAYSMPPVIKVDRPFHFMIY EETS

GMLLFLGRVVNPTLL (SEQ ID NO:25)

3. Alternative name(s): Serpin AlO

Protein Z was recently shown to act as an essential cofactor for protein Z-dependent protease inhibitor, a potent dqwnregulator of coagulation Factor Xa. Low levels of protein Z have been correlated with increased risk of stroke. However, protein Z dependent protease inhibitor was not studied. This protein has not been linked to myocardial ischemia or events leading up to MI.

G. Sodium channel suhunit beta_4

Name: ISOFORM 1 OF SODIUM CHANNEL SUBUNlT BETA-4

IPI ID: IPI00217376

UniProtKB/Swiss-Prot ID: Q8IWT1-1

Length: 228 aa, molecular weight: 24969 Da (of Precursor)

1. Basic information from UniProtKB/Swiss-Prot entry:

Modulates channel gating kinetics. Causes negative shifts in the voltage FUNCTION dependence of activation of certain alpha sodium channels, but does not affect the voltage dependence of inactivation (By similarity).

SUBCELLULAR Membrane; Single-pass type I membrane protein (Probable). LOCATION

2. Sequence:

MPGAGDGGKAPARWLGTGLLGLFLLPVTLSLEVSVGKATDIYAVNGTEILLPCTFSS CF GFEDLHFRWTYNSSDAFK]LIEGTVKNEKSDPKVTLKDDDRITLVGSTKEKMNNISIVLR DLEFSDTGKYTCHVKNPKENNLQHI IATIFLQVVDRLEEVDNTVTLIILAVVGGVTGLLIL ILLIKKLIIFILKKTREKKKECLVSSSGNDNTENGLPGSKAEEKPPSKV (SEQ ID NO:26)

This protein has not been linked to myocardial ischemia or events leading up to MI.

H. SORTILIN-RELATED RECEPTOR

Name: SORTILIN-RELATED RECEP TOR

IPI ID: IPI00022608

UniProtKB/Swiss-Prot ID: Q92673 Length: 2214 aa, molecular weight: 248441 Da (of Precursor)

1. Basic information from UniProtKB/Swiss-Prot entry:

Likely to be a multifunctional endocytic receptor, that may be implicated in the FTTNγTϊON uptake of lipoproteins and of proteases. Binds LDL, the major cholesterol- carrying lipoprotein of plasma, and transports it into cells by endocytosis. Binds the receptor- associated protein (RAP). Could play a role in cell-cell interaction.

SUBCELLULAR Membrane; Single-pass type I membrane protein (Potential). LOCATION

2. Sequence:

MATRSSRRESRLPFLFTLVALLPPGALCEVWTQRLHGGSAPLPQDRGFLVVQGDPRE LR

LWARGDARGASRADEKPLRRKRSAALQPEPIKVYGQVSLNDSHNQMVVHWAGEKSN

VIVALARDSLALARPKSSDVYVSYDYGKSFKKISDKLNFGLGNRSEAVIAQFYHSPA DN

KRYIFADAYAQYLWITFDFCNTLQGFSIPFRAADLLLHSKASNLLLGFDRSHPNKQL WK

SDDFGQTWIMIQEHVKSFSWGIDPYDKPNTIYIERHEPSGYSTVFRSTDFFQSRENQ EVIL

EEVRDFQLRDKYMFATKVVHLLGSEQOSSVOL WVSFGRKPMRAAQFVTRHPγNEYYIA

DASEDQVFVCVSHSNNRTNL YISEAEGLKFSLSLENVLYYSPGGAGSDTL VRYFANEPF

ADFHRVEGLQGVYIATLINGSMNEENMRSVITFDKGGTWEFLQAPAFTGYGEKINCE LS QGCSLHLAQRLSQLLNLQLRRMPILSKESAPGLIIATGSVGKNLASKTNVYISSSAGARW REALPGPHYYTWGDHGGIITAIAQGMFTNELKYSTNEGETWKTFIFSEKPVFVYGLLTE

PGEKSTVFTIFGSNKENVHSWLILQVNATDALGVPCTEND YKLWSPSDERGNECLLGHK

TVFKRRTPHATCFNGEDFDRPVWSNCSCTREDYECDFGFKMSEDLSLEVCVPDPEFS G

KSYSPPVPCPVGSTYRRTRGYRKISGDTCSGGDVEARLEGELVPCPLAEENEFILYA VRK

SIYR YDLASGATEQLPLTGLRAAVALDFD YEHNCLYWSDLALDVIQRLCLNGSTGQEVI

INSGLETVEALAFEPLSQLLYWVDAGFKKIEVANPDGDFRLTIVNSSVLDRPRALVL VPQ

EGVMFWTDWGDLKPGIYRSNMDGSAAYHLVSEDVKWPNGISVDDQWIYWTDAYLEC

IERITFSGQQRSVILDNLPHPYAIAVFKNEIYWDDWSQLSIFRASKYSGSQMEILAN QLTG

LMDMKIFYKGKNTGSNACVPRPCSLLCLPKANNSRSCRCPEDVSSSVLPSGDLMCDC PQ

GYQLKNNTCVKEENTCLRNQYRCSNGNCINSIWWCDFDNDCGDMSDERNCPTTICDL D

TQFRCQESGTCIPLSYKCDLEDDCGDNSDESHCEMHQCRSDEYNCSSGMCIRSSWVC D

GDNDCRDWSDEANCTAIYHTCEASNFQCRNGHCIPQRWACDGDTDCQDGSDEDPVNC

EKKCNGFRCPNGTCIPSSKHCDGLRDCSDGSDEQHCEPLCTHFMDFVCKNRQQCLFH S

MVCDGIIQCRDGSDEDAAFAGCSQDPEFHKVCDEFGFQCQNGVCISLIWKCDGMDDC G

DYSDEANCENPTEAPNCSRYFQFRCENGHCIPNRWKCDRENDCGDWSDEKDCGDSHI L

PFSTPGPSTCLPNYYRCSSGTCVMDTWVCDGYRDCADGSDEEACPLLANVTAASTPT Q

LGRCDRFEFECHQPKTCIPNWKRCDGHQDCQDGRDEANCPTHSTLTCMSREFQCEDG E

ACIVLSERCDGFLDCSDESDEKACSDELTVYKVQNLQWTADFSGDVTLTWMRPKKMP

SASCVYNVYYRVVGESIWKTLETHSNKTNTVLKVLKPDTTYQVKVQVQCLSKAHNTN

DFVTLRTPEGLPDAPRNLQLSLPREAEGVIVGHWAPPIHTHGLIREYIVEYSRSGSK MWA

SQRAASNFTEIKNLL VNTLYTVRVAA VTSRGIGNWSDSKSITTIKGKVIPPPDIHIDSYGE

NYLSFTLTMESDIKVNGYVVNLFWAFDTHKQERRTLNFRGSILSHKVGNLTAHTSYE IS

AWAKTDLGDSPLAFEHVMTRGVRPPAPSLKAKAINQTAVECTWTGPRNVVYGIFYAT S

FLDLYRNPKSLTTSLHNKTVIVSKDEQYLFLVRVVVPYQGPSSDYVVVKMIPDSRLP PR

HLHVVHTGKTSVVIKWESPYDSPDQDLLYAIAVKDLIRKTDRSYKVKSRNSTVEYTL N

KLEPGGKYHIIVQLGNMSKDSSIKITTVSLSAPDALKIITENDHVLLFWKSLALKEK HFNE

SRGYEIHMFDSAMNITAYLGNTTDNFFKISNLKMGHNYTFTVQARCLFGNQICGEPA IL

LYDELGSGADASATQAARSTDVAAVVVPILFLILLSLGVGFAILYTKHRRLQSSFTA FAN

SHYSSRLGSAIFSSGDDLGEDDEDAPMITGFSDDVPMVIA (SEQ ID NO:27)

3. Alternative name(s):

Sorting protein-related receptor containing LDLR class A repeats

Short name=SorLA SorLA-1 Low-density lipoprotein receptor relative with 11 ligand-binding repeats

Short name=LDLR relative with 11 ligand-binding repeats

Short name=LRl 1

This protein has not been linked to myocardial ischemia or events leading up to MI.

I. CONSERVED HYPOTHETIC AT, PROTEIN

Name Conserved hypothetical protein

IPI ID: 1PI00884334

Length: 168 aa, molecular weight: 18798 Da

Sequence:

MRSFLLVWKLFRRKDMKHQRKTATEFKTTEEGETRQDGKDGSLTYRADTCSPCPEAG GPPSSSIASGSSISVGNSPSHSHSHTSRRCGGSSRSRECCSSLHSSRGSRGSSWSSSPPG STC RWCSCHSHHHSHHRSHHRSHHCSHHHSHHHSGHHSHHNFHNHSNPWCQ (SEQ ID NO:28)

This protein has not been linked to myocardial ischemia or events leading up to MI.

J. CATAT,ASft

Name: Catalase

IPI ID: IPI00465436

UniProtKB/Swiss-Prot ID: P04040 Length: 527 aa, molecular weight: 59756 Da

1. Basic information from UniProtKB/Swiss-Prot entry:

Function Occurs in almost all aerobically respiring organisms and serves to protect cells from the toxic effects of hydrogen peroxide. Promotes growth of cells including T-cells, B- cells, myeloid leukemia cells, melanoma cells, mastocytoma cells and normal and transformed fibroblast cells.

Subcellular location Peroxisome.

2. Sequence:

MADSRDPASDQMQHWKEQRAAQKADVLTTGAGNPVGDKLNVITVGPRGPLLVQDVV

FTDEMAHFDRERIPERVVHAKGAGAFGYFEVTHDITKYSKAKVFEHIGKKTPIAVRF ST

VAGESGSADTVRDPRGFAVKFYTEDGNWDLVGNNTPIFFIRDPILFPSFIHSQKRNP QTH

LKDPDMVWDFWSLRPESLHQVSFLFSDRGIPDGHRHMNGYGSHTFKLVNANGEAVYC

KFHYKTDQGIKNLSVEDAARLSQEDPDYGIRDLFNAIATGKYPSWTFYIQVMTFNQA ET

FPFNPFDLTKVWPHKDYPLIPVGKLVLNRNPVNYFAEVEQIAFDPSNMPPGIEASPD KM

LQGRLFA YPDTHRHRLGPNYLHIPVNCPYRARV ANYQRDGPMCMQDNQGGAPNYYPN

SFGAPEQQPSALEHSIQYSGEVRRFNTANDDNVTQVRAFYVNVLNEEQRKRLCENI� �GH LKDAQIFIQKKAVKNFTEVHPDYGSHIQALLDKYNAEKPKNAIHTFVQSGSHLAAREKA NL (SEQ ID NO:29)

Catalase is an important enzyme in the heart's regulation ofoxidative stress. It has been linked to preconditioning in the heart tissue. As a serum marker, it has not been linked to myocardial ischemia or events leading up to MI.

K. Conserved hvnothetical protein

Name: Conserved hypothetical protein

IPI ID: IPI00883661

UniProt/TrEMBL ID: A6NFT5

Length: 175 aa, molecular weight: 20933 Da

2. Sequence:

MNIHIHTCMHIYTHAHTHAHIHTCIHTHTHMHTHTLTYTHIHMHTHTQTHIYTQAHI HS CTQINIYTYAYTLTCTQTHTHICTHAHTLTYTHIHTCTYKRTYIQGHIHTHMHTYTCTCT HTHKHIHAHIHIH I HTHIYTHTDAYTHMDTYTHTYPHTHICIHSHTHAHTYTHIRT (SEQ ID NO:30)

This protein has not been linked to myocardial ischemia or events leading up to MI.

L. Glutathione peroxidase

Name: Glutathione peroxidase 3

IPI ID: IPI00026199

UniProtKB/Swiss-Prot ID: P22352 Length: 226 aa, molecular weight: 25505 Da

1. Basic information from UniProtKB/Swiss-Prot entry:

_FUNCTION Protects cells and enzymes from oxidative damage, by catalyzing the reduction of hydrogen peroxide, lipid peroxides and organic hydroperoxide, by glutathione.

SUBCELLULAR Secreted.

LOCATION

TISSUE Secreted in plasma.

SPECIFICITY

2. Sequence:

MARLLQASCLLSLLLAGFVSQSRGQEKSKMDCHGGISGTI YEYGALTIDGEEYIPFKQY AGKYVLFVNVASYCGLTGQYIELNALQEELAPFGLVILGFPCNQFGKQEPGENSEILPTL KYVRPGGGFVPNFQLFEKGDVNGEKEQKFYTFLKNSCPPTSELLGTSDRLFWEPMKVH DIRWNFEKFLVGPDGIPIMRWHHRTTVSNVKMDILSYMRRQAALGVKRK (SEQ ID NO:31)

3. Alternative name(s): GSHPx-3

Short name=GPx-3 Extracellular glutathione peroxidase Plasma glutathione peroxidase GSHPx-P

Short name=GPx-P

GPx 3 is an important enzyme involved in many cells regulation of oxidative stress. As a serum marker it has not been linked to myocardial ischemia or events leading up to ML

M. HEPATOCYTE GROWTH FACTOR ACTIVATOR

Name: HEPATOCYTE GROWTH FACTOR ACTIVATOR

IPI ID: IPI00029193

UniProtKB/Swiss-Prot ID: Q04756

Length: 655 aa, molecular weight: 70682 Da (of Precursor)

1. Basic information from UniProtKB/Swiss-Prot entry: ci rwrτ _ϊ r» _M Activates hepatocyte growth factor (I IGF) by converting it from a single chain to a heterodimeric form.

SUBCELLULAR Secreted. Note=Secreted as an inactive single-chain precursor and is then

LOCATION activated to a heterodimeric form.

2. Sequence:

MGRWA WVPSPWPPPGLGPFLLLLLLLLLLPRGFQPQPGGNRTESPEPNATλTP AIPTILV TSVTSETP ATSAPEAEGPQSGGLPPPPRA VPSSSSPQλQALTEDGRPCRFPFRYGGRMLH ACTSEGSAHRKWCATTHNYDRDRA WGYCVEλTPPPGGPAλLDPCASGPCLNGGSCSN TQDPQSYHCSCPRAFTGKDCGTEKCFDETRYEYLEGGDRWARVRQGHVEQCECFGGR TWCEGTRHTACLSSPCLNGGTCHLIVATGTTVCACPPGFAGRLCNIEPDERCFLGNGTG YRGVASTSASGLSCLA WNSDLLYQELHVDSVGAAALLGLGPHA YCRNPDNDERPWCY VVKDSALSWEYCRLEACESLTRVQLSPDLLATLPEPASPGRQACGRRHKKRTFLRPRIIG GSSSLPGSHP WLAAIYIGDSFCAGSLVHTCWVVSAAHCFSHSPPRDSVSVVLGQHFFNR TTDVTQTFGIEKYIPYTLYSVFNPSDHDL VLIRLKKKGDRCATRSQFVQPICLPEPGSTFP AGHKCQIAGWGHLDENVSGYSSSLREALVPLVADHKCSSPEVYGADISPNMLCAGYFD CKSDACQGDSGGPLACEKNGVAYLYGI]SWGDGCGRLHKPGVYTRVANYVDWINDRI RPPRRLVAPS (SEQ ID NO:32)

This protein has not been linked to myocardial ischemia or events leading up to MI.

N. HEPATQCVTE GROWTH FACTOR-LTKE PROTFJN HOMOIX)G Name: HEPATOCYTE GROWTH FACTOR-LIKE PROTEIN HOMOLOG IPI ID: IPI00292218 UniProtKBH ^' rEMBL ID: B7Z557 Length: 697 aa, molecular weight: 78787 Da Sequence:

MLRGPCSPLNDFQVLRGTELQHLLHAVVPGPWQEDVADAEECAGRCGPLMDCRAFHY NVSSHGCQLLPWTQHSPHTRLRRSGRCDLFQKKDYVRTCIMNNGVGYRGTMATTVGG LPCQAWSHKFPNDHKYTPTLRNGLEENFCRNPDGDPGGPWCYTTDPAVRFQSCGIKSC REAACVWCNGEEYRGA VDRTESGRECQRWDLQHPHQHPFEPGKFLDQGLDDNYCRNP DGSERPWCYTTDPQIEREFCDLPRCGSEAQPRQEATTVSCFRGKGEGYRGTANTTTAGV PCQRWDAQIPHQHRFTPEKYACKDLRENFCRNPDGSEAPWCFTLRPGMRAAFCYQIRR CTDDVRPQDCYIIGAGEQYRGTVSKTRKGVQCQRWSAETPHKPQFTFTSEPHAQLEENF CRNPDGDSHGPWCYTMDPRTPFDYCALRRCADDQPPSILDPPDQVQFEKCGKRVDRLD QRRSKLRVVGGHPGNSPWTVSLRNRQGQHFCGGSLVKEQWILTARQCFSSCHMPLTGY EVWLGTLFQNPQHGEPSLQRVPVAKMVCGPSGSQL VLLKLERSVTLNQRVALICLPPE WYVVPPGTKCEIAGWGETKGTGNDTVLNV ALLNVISNQECNIKIIRGRVRESEMCTEGL LAPVGACEGDYGGPLACFTHNCWVLEGIIIPNRVCARSRWPAVFTRVSVFVDWIHKVM RLG (SEQ ID NO:33) This protein has not been linked to myocardial ischemia or events leading up to MI

O. INSUTJN-ITKE GROWTH FACTOR-RTNnINC PROTEIN 6

Name: INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN 6 IPI ID: IPI00029235 . UniProtKB/Swiss-Prot ID: P24592

Length: 240 aa, molecular weight: 25322 Da (of Precursor)

1. Basic information from UniProtKB/Swiss-Prot entry: lGF-binding proteins prolong the half-life of the IGFs and have been shown to FUNCTION either inhibit or stimulate the growth promoting effects of the IGFs on cell culture. They alter the interaction of IGFs with their cell surface receptors.

SUBCELLULAR Secreted. LOCATION

2. Sequence:

MTPHRLLPPLLLLLALLLAASPGGALARCPGCGQGVQAGCPGGCVEEEDGGSPAEGC A EAEGCLRREGQECGVYTPNCAPGLQCHPPKDDEAPLRALLLGRGRCLPARAPAVAEEN PKESKPQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGPCRRHLDSVLQQL QTEVYRGAQTLYVPNCDHRGFYRKRQCRSSQGQRRGPCWCVDRMGKSLPGSPDGNGS SSCPTGSSG (SEQ ID NO:34)

3. Synonym: IBP-6

In a swine model of myocardial injury, studied at 3-24, 72, or 168 hrs, it was shown that there was an increased level of mRNA of IGFBP-6 at all time points. In situ hybridisation identified myocytes as the main producers of IGFBP-6 mRNA. However, the protein itself was not investigated. As well, this protein was found to be elevated in a young multiple myeloma patient with high-output cardiac failure. To date, there has been no study indicating the association of this protein with myocardial ischemia or events leading up to MI.

P. Conserved hypothetical protein

Name: Conserved hypothetical protein

IPI ID: IP100847894

Length: 88 aa, molecular weight: 9931 Da

Sequence:

MFTLRLFAGKACWPVLYTMLKEVTCDVCVCVRARACTCMCMCVCECMDVCVRLYT

MLKEVTCDMCVCARTCVHVCVSAWMCVCTCTQC (SEQ ID NO:35) This protein has not been linked to myocardial ischemia or events leading up to MI.

Q. ISOFORM 1 OF RECEPTOR-TYPE TYROSINE-PROTEIN PHOSPHATASE KAPPA

Name: ISOFORM 1 OF RECEPTOR-TYPE TYROSINE-PROTEIN PHOSPHATASE KAPPA IPI ID: IPIOOOl 5756

UniProtKB/Swiss-Prot ID: Ql 5262-1

Length: 1439 aa, molecular weight: 162102 Da (of Precursor)

1. Basic information from UniProtKB/Swiss-Prot entry:

Regulation of processes involving cell contact and adhesion such as growth Fγ IMγTTπN control, tumor invasion, and metastasis. Forms complexes with beta- catenin and gamma-catenin/plakoglobin. Beta-catenin may be a substrate for the catalytic activity of PTP- kappa.

SUBCELLULAR Cell junction, adherens junction. Cell membrane; Single-pass type I LOCATION membrane protein.

2. Sequence:

MDTTAAAALPAFVALLLLSPWPLLGSAQGQFSAGGCTFDDGPGACDYHQDL YDDFEW

VHVSAQEPHYLPPEMPQGSYMIVDSSDHDPGEKARLQLPTMKENDTIICIDFSYLLY SQ

KGLNPGTLNILVRVNKGPLANPIWNVTGFTGRDWLRAELAVSTFWPNEYQVIFEAEV S

GGRSGYIAIDDIQVLSYPCDKSPIIFLRLGDVEVNAGQNATFQCIATGRDAVHNKLW LQ

RRNGEDIPVAQTKNINIIRRF AASFRLQEVTKTDQDLYRCVTQSERGSGVSNFAQLIVRE

PPRPIAPPQLLGVGPTYLLIQLNANSIIGDGPIILKEVEYRMTSGSWTETHAVMAPT YKLW

HLDPDTEYEIRVLLTRPGEGGTGLPGPPLITRTKCAEPMRTPKTLKIAEIQARRIAV DWES

LGYNITRCHTFNVTICYHYFRGI-INESKADCLDMDPKAPQHVVNHLPPYTNVSLKM ILT

NPEGRKESEETIIQTDEDVPGPVPVKSLQGTSFENKIFLNWKEPLDPNGIITQYEIS YSSIRS

FDPAVPVAGPPQTVSNLWNSTHHVFMHLHPGTTYQFFIRASTVKGFGPATAINVTTN IS

APTLPDYEGVDASLNETATTITVLLRP AQAKGAPISAYQIVVEELHPHRTKREAGAMEC

YQVPVTYQNAMSGGAPYYFAAELPPGNLPEPAPFTVGDNRTYQGFWNPPLAPRKGYN I

YFQAMSSVEKETKTQCVRJATKAATEEPEVIPDPAKQTDRVVKIAGISAGILVFILL LLVV

ILIVKKSKLAKKRKDAMGNTRQEMTHMVNAMDRSYADQSTLHAEDPLSITFMDQHNF

SPRYENHSATAESSRLLDVPRYLCEGTESPYQTGQLHPAIRV ADLLQHINLMKTSDSYGF

KEEYESFFEGQSASWDVAKKDQNRAKNRYGNIIAYDI-ISRVILQPVEDDPSSDYIN ANYI

DGYQRPSHYIATQGPVHETVYDFWRMIWQEQSACIVMVTNL VEVGRVKCYKYWPDD

TEVYGDFKVTCVEMEPLAEYVVRTFTLERRGYNEIREVKQFHFTGWPDHGVPYHATG L

LSFIRRVKLSNPPSAGPIVVHCSAGAGRTGCYIVIDIMLDMAEREGVVDIYNCVKAL RSR

RINMVQTEEQYIFIHDAILEACLCGETAIPVCEFKAAYFDMIRIDSQTNSSHLKDEF QTLN

SVTPRLQAEDCSIACLPRNHDKNRFMDMLPPDRCLPFLITIDGESSNYINAALMDSY RQP

AAFIVTQYPLPNTVKDFWRLVYDYGCTSIVMLNEVDLSQGCPQYWPEEGMLRYGPIQ V

ECMSCSMDCDVINRIFRICNLTRPQEGYLMVQQFQYLGWASHREVPGSKRSFLKLIL QV

EKWQEECEEGEGRTIIHCLNGGGRSGMFCAIGIVVEMVKRQNVVDVFHA VKTLRNSKP

NMVEAPEQYRFCYDVALEYLESS (SEQ ID NO.-36)

This protein has not been linked to myocardial ischemia or events leading up to MI.

R. TSOFORM 2 OF ATTRACTTN

Name: ISOFORM 2 OF ATTRACTIN

IPI ID: IPIOOl 62735

UniProtKB/Swiss-Prot ID: 075882-2 Length: 1272 aa, molecular weight: 141429 Da (of Precursor)

1. Basic information from UniProtKB/Swiss-Prot entry:

Involved in the initial immune cell clustering during inflammatory response and may regulate chemotactic activity of chemokines. May play a role in melanocortin FUNCTION signaling pathways that regulate energy homeostasis and hair color. Low-affinity receptor for agouti (By similarity). Has a critical role in normal myelination in the central nervous system (By similarity).

SUBCELLULAR Secreted. LOCATION

2. Sequence:

MVAAAAATEARLRRRTAATAALAGRSGGPHWDWDVTRAGRPGLGAGLRLPRLLSPPL

RPRLLLLLLLLSPPLLLLLLPCEAEAAAAAAAVSGSAAAEAKECDRPCVNGGRCNPG TG

QCVCPAGWVGEQCQHCGGRFRLTGSSGFVTDGPGNYKYKTKCTWLIEGQPNRIMRLR

FNHFATECSWDHLYVYDGDSIYAPLVAAFSGLIVPERDGNETVPEVVATSGYALLHF FS

DAAYNLTGFNITYSFDMCPNNCSGRGECKISNSSDTVECECSENWKGEACDIPHCTD NC

GFPHRGICNSSDVRGCSCFSDWQGPGCSVPVPANQSFWTREEYSNLKLPRASHKAVV N

GNIMWVVGGYMFNHSDYNMVLAYDLASREWLPLNRSVNTMVVVRYGHSLALYKDKIY

MYGGKIDSTGNVTNELRVFHIHNESWVLLTPKAKEQYAVVGHSAHIVTLKNGRVVML

VIFGHCPL YGYISNVQEYDLDKNTWSILHTQGAL VQGGYGHSSVYDHR TRALYVHGGY

KAFSANKYRLADDLYRYDVDTQMWTILKDSRFFRYLHTAVIVSGTMLVFGGNTHNDT

SMSγIGAKCFSSDFMAYDIACDRWSVLPRPDLHHDVNRFGHSAVLHNSTMYVFGGF NS

LLLSDILVFTSEQCDAHRSEAλCLAAGPGIRCVWNTGSSQCISWALATDEQEEKLK SECF

SKRTLDHDRCDQHTDCYSCTANTNDCHWCNDHCVPRNHSCSEGQISIFRYENCPKDN P

MYYCNKKTSCRSCALDQNCQWEPRNQECIALPENICGIGWHLVGNSCLKITTAKENY D

NAKLFCRNHNALLASLTTQKKVEFVLKQLRIMQSSQSMSKLTLTPWVGLRKINVSYW C

WEDMSPFTNSLLQWMPSEPSDAGFCGILSEPSTRGLKAATCINPLNGSVCERPANHS AK

QCRTPCALRTACGDCTSGSSECMWCSNMKQCVDSNAYVASFPFGQCMEWYTMSTCPP

ENCSGYCTCSHCLEQPGCGWCTDPSNTGKGKCIEGSYKGPVKMPSQAPTGNFYPQPL L

NSSMCLEDSRYNWSFIHCPACQCNGHSKCINQSICEKCENLTTGKHCETCISGFYGD PTN

GGKCQPCKCNGHASLCNTNTGKCFCTTKGVKGDECQLCEVENRYQGNPLRGTCYYTL

LIDYQFTFSLSQEDDRYYTAINFVATPDEQNRDLDMFINASKNFNLNITWAASFSAG TQ

AGEEMPVVSKTNIKEYKDSFSNEKFDFRNHPNITFFVYVSNFTWPIKIQVQTEQ (SEQ ID

NO:37)

3. Alternative name(s): Mahogany homolog; DPPT-L

This protein has not been linked to myocardial ischemia and events leading up to MI.

S. ISOFORM A OF SVNTAXIN-3

Name: Syntaxin-3 (STX3A)

IPI ID: IPI00395768

UniProtKB/Swiss-Prot ID: Q 13277-1 and Ql 3277-2

Length: 289 aa, molecular weight: 33155 Da Q13277-1

1. Basic information from UniProtKB/Swiss-Prot entry:

FUNCTION Potentially involved in docking of synaptic vesicles at presynaptic active zones.

SUBCELLULAR Membrane; Single-pass type IV membrane protein (Potential).

LOCATION

2. Sequence:

MKDRLEQLKAKQLTQDDDTDAVEIAIDNTAFMDEFFSEIEETRLNIDKISEHVEEAK KLY SHLSAPIPEPKTKDDLEQLTTEIKKRANNVRNKLKSMEKHIEEDEVRSSADLRIRKSQHS VLSRKFVEVMTKYNEAQVDFRERSKGRIQRQLEITGKKTTDEELEEMLESGNPAIFTSGI IDSQISKQALSEIEGRHKDγVRLESSIKELHDMFMDIAML VENQGEMLDNIELNVMHTVD HVEKARDETKKAVKYQSQARKKLIIIIVLVVVLLGILALIIGLSVGLN (SEQ ID NO: 38)

This protein has not been linked to myocardial ischemia and events leading up to MI.

T. Laetotransferjrjn

Name: Lactotransferrin

IPI ID: IPI00789477

UniProtKB/TrEMBL ID: B2MV14. B7Z4X2 Length: 666 aa, molecular weight: 73161 Da

Sequence:

MRKVRGPPVSCIKRDSPIQCIQAIλENRADA VTLDGGFIYEAGLAP YKLRPVλAEVYGT ERQPRTHYYAVAVVKKGGSFQLNELQGLKSCHTGLRRTAGWNVPIGTLRPFLNWTGPP EPLEAA VARFFSASCVPGADKGQFPNLCRLCAGTGENKCAFSSQEPYFSYSGAFKCLRD GAGDVAFIRESTVFEDLSDEAERDEYELLCPDNTRKPVDKFKDCHLARVPSHAVVARS VNGKEDAIWNLLRQAQEKFGKDKSPKFQLFGSPSGQKDLLFKDSAIGFSRVPPRIDSGL YLGSGYFTAIQNLRKSEEEVAARRARVVWCA VGEQELRKCNQWSGLSEGSVTCSSAST TEDCIALVLKGEAD AMSLDGGYV YTAGKCGLVPVLAENYKSQQSSDPDPNCVDRPVE GYLA VAVVRRSDTSLTWNSVKGKKSCHTA VDRTAGWNIPMGLLFNQTGSCKFDEYFS QSCAPGSDPRSNLCALCIGDEQGENKCVPNSNERYYGYTGAFRCLAENAGDVAFVKDV TVLQNTDGNNNEA WAKDLKLADFALLCLDGKRKPVTEARSCHLAMAPNHAVVSRMD KVERLKQVLLHQQAKFGRNGSDCPDKFCLFQSETKNLLFNDNTECLARLHGKTTYEKY LGPQYVAGITNLKKCSTSPLLEACEFLRK (SEQ ID NO.-39)

Levels have been shown to increase with leukocyte activation. Therefore, there are increases found during ischemic stroke, following by-pass surgery and after direct stenting in patients with angina. However, no studies that have linked this protein to myocardial ischemia or events leading up to MI.

UJLQE-JPRNSITY LIPOPROTEIN RECEPTOR-RELATED PROTETN 2

Name: LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN 2

IPl ID: IPI00024292

UniProtKB/Swiss-Prόt ID: P98164

Length: 4655 aa, molecular weight: 521958 Da (of Precursor)

1. Basic information from UniProtKB/Swiss-Prot entry:

Acts together with ciibilin to mediate HDL endocytosis (By similarity). May FUNCTION participate in regulation of parathyroid- hormone and para-thyroid-hormone- related protein release.

SUBCELLULAR Membrane; Single-pass type I membrane protein. Membrane, coated pit. LOCATION

2. Sequence:

MDRGPAAVACTLLLALVACLAPASGQECDSAHFRCGSGHCIPADWRCDGTKDCSDDA

DEIGCAWTCQQGYFKCQSEGQCIPNSWVCDQDQDCDDGSDERQDCSQSTCSSHQITC S

NGQCIPSEYRCDHVRDCPDGADENDCQYPTCEQLTCDNGACYNTSQKCDWKVDCRDS

SDEINCTEICLHNEFSCGNGECIPRAYVCDHDNDCQDGSDEHACNYPTCGGYQFTCP SG

RCIYQNWVCDGEDDCKDNGDEDGCESGPHDVHKCSPREWSCPESGRCISIYKVCDGI L

DCPGREDENNTSTGKYCSMTLCSALNCQYQCHETPYGGACFCPPGYIINHNDSRTCV EF

DDCQIWGICDQKCKSRPGRHLCHCEEGYILERGQYCKANDSFGEASIIFSNGRDLLI GDI

HGRSFRILVESQNRGVAVGVAFHYHLQRVFWTDTVQNKVFSVDINGLNIQEVLNVSV E TPENLAVDWVNNKIYLVETKVNRIDMVNLDGSYRVTLITENLGHPRGIAVDPTVGYLFF SDWESLSGEPKLERAFMDGSNRKDLVKTKLGWPAGVTLDMISKRVYWVDSRFDYIET VTYDGIQRKTVVHGGSLIPHPFGVSLFEGQVFFTDWTKMAVLKANKFTETNPQVYYQA SLRPYGVTVYHSLRQPYATNPCKDNNGGCEQVCVLSHRTDNDGLGFRCKCTFGFQLDT DERHCIAVQNFLIFSSQVAIRGIPFTLSTQEDVMVPVSGNPSFFVGIDFDAQDSTIFFSD MS KHMIFKQKIDGTGREILAANRVENVESLAFDWISKNLYWTDSHYKSISVMRLADKTRRT VVQYLNNPRSVVVHPFAGYLFFTDWFRPAKIMRAWSDGSHLLPVINTTLGWPNGLAID WAASRI. YWVDAYFDKIEHSTFDGLDRRRLGHIEQMTHPFGLAIFGEHLFFTD WRLGAII RVRKADGGEMTVIRSGIA YILHLKS YD VNIQTGSN ACNQPTHPNGDCSHFCFPVPNFQR VCGCPYGMRLASNHLTCEGDPTNEPPTEQCGLFSFPCKNGRCVPNYYLCDGVDDCHDN SDEQLCGTLNNTCSSSAFTCGHGECIPAHWRCDKRNDCVDGSDEHNCPTHAPASCLDT QYTCDNHQCISKNWVCDTDNDCGDGSDEKNCNSTETCQPSQFNCPNHRCIDLSFVCDG DKDCVDGSDEVGCVLNCTASQFKCASGDKCIGVTNRCDGVFDCSDNSDEAGCPTRPPG MCHSDEFQCQEDGICIPNFWECDGHPDCLYGSDEIINACVPKTCPSSYFHCDNGNCIHRA WLCDRDNDCGDMSDEKDCPTQPFRCPSWQWQCLGHNICVNLSVVCDGIFDCPNGTDE SPLCNGNSCSDFNGGCTHECVQEPFGAKCLCPLGFLLANDSKTCEDIDECDILGSCSQHC YNMRGSFRCSCDTGYMLESDGRTCKVTASESLLLLVASQNKIIADSVTSQVHNIYSLVE NGSYIVAVDFDSISGRIFWSDATQGKTWSAFQNGTDRRWFDSSIILTETIAID WVGRNL YWTDYALETI EVSKIDGSHRTVLISKNLTNPRGLALDPRMNEHLLFWSDWGHHPRIERA SMDGSMRTVIVQDKIFWPCGLTIDYPNRLL YFMDSYLDYMDFCDYNGHHRRQVIASDL IIRHPYALTLFEDSVYWTDRATRRVMRANKWIIGGNQSVVMYNIQWPLGIVAVHPSKQ PNSVNPCAFSRCSHLCLLSSQGPHFYSCVCPSGWSLSPDLLNCLRDDQPFLITVRQHIIF GI SLNPEVKSNDAMVPIAGIQNGLDVEFDDAEQYIYWVENPGEIHRVKTDGTNRTVFASIS MVGPSMNLALDWISRNLYSTNPRTQSIEVLTLHGDIRYRKTLIANDGTALGVGFPIGITV DPARGKLYWSDQGTDSGVPλKIASANMDGTSVKTLFTGNLEHLECVTLDIEEQKLYWA VTGRGVIERGNVDGTDRMILVHQLSHPWGIAVHDSFLYYTDEQYEVIERVDKATGANK IVLRDNVPNLRGLQVYHRRNAAESSNGCSNNMNACQQICLPVPGGLFSCACATGFKLN PDNRSCSPYNSFIVVSMLSAIRGFSLELSDHSETMVPVAGQGRNALHVDVDVSSGFIYW CDFSSSVASDNAIRRIKPDGSSLMNIVTHGIGENGVRGIAVDWVAGNLYFTNAFVSETLI EVLRINTTYRRVLLKVTVDMPRHIVVDPKNRYLFWADYGQRPKIERSFLDCTNRTVLVS EGIVTPRGLA VDRSDGYVYWVDDSLDIIARIRINGENSEVIRYGSR YPTPYGITVFENSII WVDRNLKKIFQλSKEPENTEPPTVIRDNINWLRDVTIFDKQVQPRSPAEVNNNPCLENN GGCSHLCFALPGLHTPKCDCAFGTLQSDGKNCλISTENFLIFALSN SLRS LHLDPENHSPP FQTINVERTVMSLDYDSVSDRIYFTQNLASGVGQISYATLSSGIHTPTVIASGIGTADGI A FDWπ RRIYYSDYLNQMINSMAEDGSNRTVIARVPKPRλIVLDPCQGYLYWADWDTHA KIERATLGGNFRVPIVNSSLVMPSGLTLDYEEDLLYWVDASLQRIERSTLTGVDREVIVN AAVHAFGLTL YGQ YL YWTDL YTQRIYRANKYDGSGQIAMTTNLLSQPRGINTVVKNQK QQCNNPCEQFNGGCSHICAPGPNGAECQCPHEGNWYLANNRKHCIVDNGERCGASSFT CSNGRCISEEWKCDNDNDCGDGSDEMESVCALHTCSPTAFTCANGRCVQYSYRCDYY NDCGDGSDEAGCLFRDCNATTEFMCNNRRCIPREFICNGVDNCHDNNTSDEKNCPDRT CQSGYTKCHNSNICIPRVYLCDGDNDCGDNSDENPTYCTTHTCSSSEFQCASGRCIPQH WYCDQETDCFDASDEPASCGHSERTCLADEFKCDGGRCIPSEWICDGDNDCGDMSDED KRHQCQNQNCSDSEFLCVNDRPPDRRCIPQSWVCDGDVDCTDGYDENQNCTRRTCSE NEFTCGYGLCIPKIFRCDRHNDCGDYSDERGCLYQTCQQNQFTCQNGRCISKTFVCDED NDCGDGSDELMHLCHTPEPTCPPHEFKCDNGRCIEMMKLCNHLDDCLDNSDEKGCGIN ECHDPSISGCDHNCTDTLTSFYCSCRPGYKLMSDKRTCVDIDECTEMPFVCSQKCENVI GSYICKCAPGYLREPDGKTCRQNSNIEPYLIFSNRYYLRNLTIDGYFYSLILEGLDNVVA L DFDRVEKRLYWIDTQRQVIERMFLNKTNKETIINHRLPAAESLAVDWVSRKLYWLDAR LDGLFVSDLNGGIIRRMLAQHCVDANNTFCFDNPRGLALHPQYGYLYWADWGHRAYI

GRVGMDGTNKSVIISTKLEWPNGITIDYTNDLLYWADAHLGYIEYSDLEGHHRHTVY D

GALPHPFAITIFEDTIYWTDWNTRTVEKGNKYDGSNRQTLVNTTHRPFDIHVYHPYR QPI

VSNPCGTNNGGCSHLCLIKPGGKGFTCECPDDFRTLQLSGSTYCMPMCSSTQFLCAN NE

KCIPIWWKCDGQKDCSDGSDELALCPQRFCRLGQFQCSDGNCTSPQTLCNAHQNCPD G

SDEDRLLCENHHCDSNEWQCANKRCIPESWQCDTFNDCEDNSDEDSSHCASRTCRPG Q

FRCANGRCIPQAWKCDVDNDCGDHSDEPIEECMSSAHLCDNFTEFSCKTNYRCIPKW A

VCNGVDDCRDNSDEQGCEERTCHPVGDFRCKNHHCIPLRWQCDGQNDCGDNSDEENC

APRECTESEFRCVNQQCIPSRWICDHYNDCGDNSDERDCEMRTCHPEYFQCTSGHCV HS

ELKCDGSADCLDASDEADCPTRFPDGAYCQATMFECKNHVCIPPYWKCDGDDDCGDG

SDEELHLCLDVPCNSPNRFRCDNNRCIYSHEVCNGVDDCGDGTDETEEHCRKPTPKP CT

EYEYKCGNGHCIPHDNVCDDADDCGDWSDELGCNKGKERTCAENICEQNCTQLNEGG

FICSCTAGFETNVFDRTSCLDINECEQFGTCPQHCRNTKGSYECVCADGFTSMSDRP GK

RCAAEGSSPLLLLPDNVRIRKYNLSSERFSEYLQDEEYIQA VD YDWDPKDIGLSVVYYT

VRGEGSRFGAIKRAYIPNFESGRNNLVQEVDLKLKYVMQPDGIAVDWVGRHIYWSDV

KNKRIEVAKLDGRYRKWLISTDLDQPAAIAVNPKLGLMFWTDWGKEPKIESAWMNGE

DRNILVFEDLGWPTGLSIDYLNNDRIYWSDFKEDVIETIKYDGTDRRVIAKEAMNPY SL

DIFEDQLYWISKEKGEVWKQNKFGQGKKEKTLVVNPWLTQVRIFHQLRYNKS VPNLC

KQICSHLCLLRPGGYSCACPQGSSFIEGSTTECDAAIELPINLPPPCRCMHGGNCYF DETD

LPKCKCPSGYTGKYCEMAFSKGISPGTTAV AVLLTILLIVVIGALAIAGFFHYRRTGSLLP

ALPKLPSLSSLVKPSENGNGVTFRSGADLNMDIGVSGFGPETAIDRSMAMSEDFVME M

GKQPIIFENPMYSARDSAVKWQPIQVTVSENVDNKNYGSPINPSEIVPETNPTSPAA DG

TQVTKWNLFKRKSKQTTNFENPIYAQMENEQKESVAATPPPSPSLPAKPKPPSRRDP TPT

YSATEDTFKDTANLVKEDSEV (SEQ ID NO:40)

3. Alternative name(s): Megalin; Glycoprotein 330; Short name=gp330

This protien has not been directly linked to myocardial ischemia or events leading up Io MI.

V. Pro I QW density lipoprotein receptor related protein 1

Name: Prolow density lipoprotein receptor related protein 1

IPI ID: IPI00020557 ^•

UniProtKB/Swiss-Prot ID: Q07954

Length: 4544 aa, molecular weight: 504575 Da (of Precursor)

1. Basic information from UniProtKB/Swiss-Prot entry:

• Function: Endocytic receptor involved in endocytosis and in phagocytosis of apoptotic cells. Required for early embryonic development. Involved in cellular lipid homeostasis. Involved in the plasma clearance of chylomicron remnants and activated LRPAPl (alpha 2-macroglobulin), as well as the local metabolism of complexes between plasminogen activators and their endogenous inhibitors. May modulate cellular events, such as APP metabolism, kinase-dependent intracellular signaling, neuronal calcium signaling as well as neurotransmission.

• Subcellular location: Low-density lipoprotein receptor-related protein 1 85 kDa subunit: Cell membrane; Single-pass type I membrane protein. Membrane > coated pit. Low-density lipoprotein receptor-related protein 1 515 kDa subunit: Cell membrane; Peripheral membrane protein; Extracellular side. Membrane > coated pit. Low-density lipoprotein receptor-related protein 1 intracellular domain: Cytoplasm. Nucleus. Note= After cleavage, the intracellular domain (LRPICD) is detected both in the cytoplasm and in the nucleus.

2. Sequence:

MLTPPLLLLLPLLSALVAAAIDAPKTCSPKQFACRDQITCISKGWRCDGERDCPDGS DEA

PEICPQSKAQRCQPNEHNCLGTELCVPMSRLCNGVQDCMDGSDEGPHCRELQGNCSR LGC

QHHCVPTLDGPTCYCNSSFQLQADGKTCKDFDECSVYGTCSQLCTNTDGSFICGCVE GYL

LQPDNRSCKAKNEPVDRPPVLLIANSQNILATYLSGAQVSTITPTSTRQTTAMDFSY ANE

TVCWVHVGDSAAQTQLKCARMPGLKGFVDEHTγNISLSLHHVEQMAIDWLTGNFYF VDDI

DDRIFVCNRNGDTCVTLLDLEL YNPKGIALDPAMGKVFFTDYGQIPKVERCDMDGQNRTK

LVDSKIVFPHGITLDLVSRLVYWADAYLDYIEVVDYEGKGRQTIIQGILIEHLYGLT VFE

NYLY ATNSDNANAQQKTSVIRVNRFNSTEYQVVTRVDKGGALHIYHQRRQPRVRSHACEN

DQYGKPGGCSDICLLANSHKARTCRCRSGFSLGSDGKSCKKPEHELFLVYGKGRPGI IRG

MDMGAKVPDEHMIPIENLMNPRALDFHAETGFIYFADTTSYLIGRQKIDGTERETIL KDG

IHNVEGVAVDWMGDNLYWTDDGPKKTISVARLEKAAQTRKTLIEGKMTHPRAIWDPL NG

WMYWTDWEEDPKDSRRGRLERAWMDGSHRDIFVTSK'I VLWPNGLSLDIPAGRLYWVDAFY

DRIETILLNGTDRKIVYEGPELNHAFGLCHHGNYLFWTEYRSGSVYRLERGVGGAPP TVT

LLRSERPPIFEIRMYDAQQQQVGTNKCRVNNGGCSSLCLATPGSRQCACAEDQVLDA DGV

TCLANPSYVPPPQCQPGEFACANSRCIQERWKCDGDNDCLDNSDEAPALCHQHTCPS DRF

KCENNRCIPNRWLCDGDNDCGNSEDESNATCSARTCPPNQFSCASGRCIPISWTCDL DDD

CGDRSDESASCAYPTCFPLTQFTCNNGRCININWRCDNDNDCGDNSDEAGCSHSCSS TQF

KCNSGRCIPEHWTCDGDNDCGDYSDETHANCTNQATRPPGGCHTDEFQCRLDGLCIP LRW

RCDGDTDCMDSSDEKSCEGVTHVCDPSVKFGCKDSARCISKAWVCDGDNDCEDNSDE ENC

ESLACRPPSHPCANNTSVCLPPDKLCDGNDDCGDGSDEGELCDQCSLNNGGCSHNCS VAP

GEGIVCSCPLGMELGPDNHTCQIQSYCAKHLKCSQKCDQNKFSVKCSCYEGWVLEPD GES

CRSLDPFKPFIIFSNRHEIRRIDLHKGDYSVLVPGLRNTIALDFHLSQSALYWTDVV EDK

IYRGKLLDNGALTSFEVVIQYGLATPEGLA VDWIAGNIYWVESNLDQIEVAKLDGTLRTT

LLAGDIEHPRAIALDPRDGTLFWTDWDASLPRIEAASMSGAGRRTVHRETGSGGWPN GLT

VDYLEKRILWIDARSDAIYSARYDGSGHMEVLRGHEFLSHPFAVTLYGGEVYWTDWR TNT

LAKANKWTGIINVTVVQRTNTQPFDLQVYHPSRQPMAPNPCEANGGQGPCSHLCLIN YNRT

VSCACPHLMKLHKDNTTCYEFKKFLL YλRQMEIRGVDLDAPYYNYIISFTVPDIDNVTVL

DYDAREQRVYWSDVRTQλIKRAFINGTGVETVVSADLPNAHGLAVDWVSRNLFWTS YDTN

KKQINVλRLDGSFKNAVVQGLEQPHGLVVHPLRGKLYWTDGDNISMANMDGSNRTL LFSG

QKGPVGLAIDFPESKLYWISSGNHTINRCNLDGSGLEVIDAMRSQLGKATALAIMGD KLW

WADQVSEKMGTCSKADGSGSVVLRNSTTLVMHMKVYDESIQLDHKGTNPCSVNNGDC SQL

CLPTSETTRSCMCTAGYSLRSGQQACEGVGSFLLYSVHEGIRGIPLDPNDKSDALVP VSG

TSLAVGIDFHAENDTIYWVDMGLSTISRAKRDQTWREDVVTNGIGRVEGIAVDWIAG NIY

WTDQGFDVIEVARLNGSFRYVVISQGLDKPRAITVHPEKGYLFWTEWGQYPRIERSR LDG

TERVVLVNVSISWPNGISVDYQDGKLYWCDARTDKIERIDLETGENREVVLSSNNMD MFS

VSVFEDFIYWSDRTHANGSIKRGSKDNATDSVPLRTGIGVQLKDIKVFNRDRQKGTN VCA

VANGGCQQLCLYRGRGQRACACAHGMLAEDGASCREYAGYLLYSERTILKSIHLSDE RNL

NAPVQPFEDPEHMKNVIALAFDYRAGTSPGTPNRIFFSDIHFGNIQQINDDGSRRIT IVE

NVGSVEGLλYHRGWDTLYWTSYTTSTITRHTVDQTRPGAFERETVITMSGDDHPRA FVLD

ECQNLMFWTN WNEQHPSIMRAALSGANVLTLIEKDIRTPNGLAIDHRAEKLYFSDATLDK

IERCEYDGSHRYVILKSEPVHPFGLAVYGEHIFWTD WVRRAVQRANKHVGSNMKLLRVDI

PQQPMGIIA VANDTNSCELSPCRINNGGCQDLCLLTHQGIIVNCSCRGGRILQDDLTCRAV

NSSCRAQDEFECANGECγNFSLTCDGVPHCKDKSDEKPSYCNSRRCKKTFRQCSNG RCVS

NMLWCNGADDCGDGSDEIPCNKTACGVGEFRCRDGTCIGNSSRCNQFVDCEDASDEM NCS

ATDCSSYFRLGVKGVLFQPCERTSLCYAPSWVCDGANDCGDYSDERDCPGVKRPRCP LNY

FACPSGRCIPMSWTCDKEDDCEHGEDETHCNKFCSEλQFECQNHRCISKQWLCDGS DDCG

DGSDEλAHCEGKTCGPSSFSCPGTHVCVPERWLCDGDKDCADGADESIAAGCLYNS TCDD

REFMCQNRQCIPKHFVCDHDRDCADGSDESPECEYPTCGPSEFRCANGRCLSSRQWE CDG

ENDCHDQSDEAPKNPHCTSPEHKCNASSQFLCSSGRCVAEALLCNGQDDCGDSSDER GCH

INECLSRKLSGCSQDCEDLKIGFKCRCRPGFRLKDDGRTCADVDECSTTFPCSQRCI NTH

GSYKCLCVEGYAPRGGDPHSCKA VTDEEPFLIFANRYYLRKLNLDGSNYTLLKQGLNNAV

ALDFDYREQMIYWTDVTTQGSMIRRMHLNGSNVQVLHRTGLSNPDGLA VDWVGGNLYWCD KGRDTϊEVSKLNGA YRTVLVSSGLREPRALVVDVQNGYLYWTDWGDHSLIGRIGMDGSSR SVIVDTKITWPNGLTLDYVTERJYWADAREDYIEFASLDGSNRHVVLSQDIPHIFALTLF EDYVYWTDWETKSINRAHKTTGTNKTLLISTLHRPMDLHVFHALRQPDVPNHPCKVNNGG CSNLCLLSPGGGHKCACPTNFYLGSDGRTCVSNCTASQFVCKNDKCIPFWWKCDTEDDCG DHSDEPPDCPEFKCRPGQFQCSTGICTNPAFICDGDNDCQDNSDEANCDIHVCLPSQFKC TNTNRCIPGIFRCNGQDNCGDGEDERDCPEVTCAPNQFQCSITKRCIPRVWVCDRDNDCV DGSDEPANCTQMTCGVDEFRCKDSGRC]PARWKCDGEDDCGDGSDEPKEECDERTCEPYQ FRCKNNRCVPGRWQCDYDNDCGDNSDEESCTPRPCSESEFSCANGRCIAGRWKCDGDHDC ADGSDEKDCTPRCDMDQFQCKSGHCIPLRWRCDADADCMDGSDEEACGTGVRTCPLDEFQ CNNTLCKPLAWKCDGEDDCGDNSDENPEECARFVCPPNRPFRCKNDRVCLWIGRQCDGTD NCGDGTDEEDCEPPTλHTTHCKDKKEFLCRNQRCLSSSLRCNMFDDCGDGSDEEDCSID P KLTSCATNASICGDEARCVRTEKAAYCACRSGFHTVPGQPGCQDINECLRFGTCSQLCNN TKGGHLCSCARNFMKTHNTCKAEGSEYQVLY]ADDNEIRSLFPGHPHSAYEQAFQGDESV RIDAMDVHVKAGRVYWTNWHTGTISYRSLPPAAPPTTSNRHRRQIDRGVTHLNISGLKMP RGIAIDWV AGNVYWTDSGRD VIEVAQMKGENRKTLISGMIDEPHAIVVDPLRGTMYWSDW GNHPKIETAAMDGTLRETLVQDNIQWPTGLAVDYHNERLYWADAKLSVIGSIRLNGTDPI VAADSKRGLSHPFSIDVFEDYIYGVTYINNRVFKIHKFGHSPLVNLTGGLSHASDVVLYH QHKQPEVTNPCDRKKCEWLCLLSPSGPVCTCPNGKRLDNGTCVPVPSPTPPPDAPRPGTC NLQCFNGGSCFLNARRQPKCRCQPRYTGDKCELDQCWEHCRNGGTCAASPSGMPTCRCPT GFTGPKCTQQVCAGYCANNSTCTVNQGNQPQCRCLPGFLGDRCQYRQCSGYCENFGTCQM AADGSRQCRCTAYFEGSRCEVNKCSRCLEGACVVNKQSGDVTCNCTDGRVAPSCLTCVGH CSNGGSCTMNSKMMPECQCPPHMTGPRCEEHVFSQQQPGHIASILIPLLLLLLLVLVAGV VFWYKItRVQGAKGFQHQRMTNGAMNVEIGNPTYKMYEGGEPDDVGGLLDADFALDPDKP T NFTNPVYATLYMGGHGSRHSLASTDEKRELLGRGPEDEIGDPLA (SEQ ID NO:41)

3. Alternative name(s): Alpha-2-macroglobulin receptor

Short name=A2MR Apolipoprotein E receptor

Short name=APOER CD_antigen=CD91

This protien has not been directly linked to myocardial ischemia or events leading up to Ml.

W. MONOCYTE DIFFERENTIATION ANTIGEN CD14

Name: MONOCYTE DIFFERENTIATION ANTIGEN CD 14

IPI ID: IPI00029260

UniProtKB/Swiss-Prot ID: P08571 Length: 375 aa, molecular weight: 40076 Da (of Precursor)

1. Basic information from UniProtKB/Swiss-Prot entry:

Cooperates with MD-2 and TLR4 to mediate the innate immune response to

FUNCTION bacterial lipopolysaccharide (LPS). Acts via MyD88, TIRAP and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response. Up- regulates cell surface molecules, including adhesion molecules.

SUBCELLULAR Cell membrane; Lipid-anchor, GPl-anchor. LOCATION

2. Sequence: MERASCLLLLLLPLVHVSATTPEPCELDDEDFRCVCNFSEPQPDWSEAFQCVSAVEVEIH AGGLNLEPFLKRVDADADPRQYADTVKALRVRRLTVGAAQVPAQLLVGALRVLAYSR

LKELTLEDLKITGTMPPLPLEATGLALSSLRLRNVSWATGRSWLAELQQWLKPGLKV LS IAQAHSPAFSCEQVRAFPALTSLDLSDNPGLGERGLMAALCPHKFPAIQNLALRNTGME TPTGVCAALAAAGVQPHSLDLSHNSLRATVNPSAPRCMWSSALNSLNLSFAGLEQVPK GLPAKLRVLDLSCNRLNRAPQPDELPEVDNLTLDGNPFLVPGTALPHEGSMNSGVVPA 5 CARSTLSVGVSGTLVLLQGARGFA (SEQ ID NO:42)

3. Alternative name(s): Myeloid cell-specific leucine-rich glycoprotein; CD_antigcn=CD14

Monocytes and T-cells play an important role in the development of atherosclerotic coronary 10 artery disease. Cl 4 is located on the monocytes and, therefore, changes to this protein can and have been linked to alterations to monocytes (including with coronary artery disease). However, this protein has not been measured in serum in context to myocardial ischemia or events leading up to a MI.

5 X. Peroxiredoxin-2 ,

Name: Peroxiredoxin-2 IPI ID: IPI00027350

UniProtKB/Swiss-Prot ID: P32119; PRDX2JIUMAN; M.

Length: 198 aa, molecular weight: 21892 Da, CRC64 checksum: 1AC781D908B32B46 !0

1. Basic information from UniProtKB/Swiss-Prot entry:

Function Involved in redox regulation of the cell. Reduces peroxides with reducing equivalents provided through the thioredoxin system. It is not able to receive electrons from glutaredoxin. May play an important role in eliminating peroxides generated during metabolism. Might participate in the signaling cascades of growth factors and tumor necrosis factor-alpha by regulating the intracellular concentrations OfH ₂ O ₂ .

Catalytic activity 2 R'-SH + ROOH = R'-S-S-R ¹ + H ₂ O + ROH.

Subunit structure Homodimer; disulfide-linked, upon oxidation. May be found as a toroid-shaped decamer composed of 5 dimers, depending on pH and calcium concentration. Interacts with TlPIN.

Subcellular location Cytoplasm.

Miscellaneous The active site is the redox-active Cys-51 oxidized to Cys-

SOH. Cys-SOH rapidly reacts with Cys-172-SH of the other subunit to form an intermolecular disulfide with a concomitant homodimer formation. The enzyme may be subsequently regenerated by reduction of the disulfide by thioredoxin.

Inactivated upon oxidative stress by overoxidation of Cys- 51 to CyS-SO ₂ H and CyS-SO ₃ H. CyS-SO ₂ H is retroreduced to Cys-SOH after removal OfH ₂ O ₂ , while CyS-SO ₃ H may be irreversibly oxidized.

Sequence similarities Belongs to the ahpC/TSA family.

Contains 1 thioredoxin domain.

2. Sequence:

MASGNARIGKPAPDFKATλ VVDGAFKEVKLSD YKGKYVVLFFYPLDFTFVCPTEϊI AFS NRAEDFRKLGCEVLGVSVDSQFTHLAWINTPRKEGGLGPLNIPLLADVTRRLSEDYGVL KTDEGIAYRGLFIIDGKGVLRQITVNDLPVGRSVDEALRLVQAFQYTDEHGEVCPAGW KPGSDTIKPNVDDSKEYFSKHN (SEQ ID NO:43)

3. Alternative name(s): Thioredoxin peroxidase 1 Thioredoxin-dependent peroxide reductase 1

Thiol-specifϊc antioxidant protein

Short name=TSλ PRP

Natural killer cell-enhancing factor B Short name=NKEF-B

This protein has been found to increase in the serum of a number diseases but none are cardiac related. This protein, to date, has not been shown to be increased in myocardial ischemia or events leading to MI.

Y. NCOR2 CTG26 alternate open readinp; frame Name: CTG26 alternate open reading frame (Fragment) IPI ID: IPI00006659.3 1. Basic information: Fragment

2. Sequence:

SFSSMEASSALCWGVMASSLLASLAIERVMRPLRLPWLLAVLRPLEATASFSSLSSP EVS SVFSLRRSSLSFSTSGFSSSFSASFSFSFSSFSSWLLRGMGCCCCCCCCCCCCCCCCWLL P RRR (SEQ ID NO:44)

This protein has not be linked to myocardial ischemia or events leading up to MI.

Example V. Validation studies

Antibodies to two or more epitopes on each protein will be generated and used to develop a sandwich ELISA assay (as single or multiplex) that is specific and senstive for the analyte. The analyte will either be peptide, protein fragement or protein and will be used to generate standard curve. Analyiss will be carried out using conventional ELISA or on a Luminex or Mesoscale platform. Assays will be carried out at least in duplicate. For MRM assays, peptides (generated most likely by trypsin, chymotrypsin or Lys C) that are unique to the protein of interest and showing high MS signal response (prototypic peptides) which will help maximize the sensitivity of the assay. 2. Selection of predominant peptide fragments specific (MS/MS) for the parent peptide (useful MRM transition). 3. For each peptide-fragment pair, optimization of specific MS parameters (e.g. the collision energy) to maximize the signal

response/sensitivity. 4. Validation of the MRM assay to confirm, peptide identity, e.g. by acquiring a full MS2 spectrum of the peptide in the triple quadrupole MS instrument used for MRM. 5. Extraction of the final "coordinates" of the MRM assay, including the selected peptide and peptide fragments, the corresponding mass-to-charge ratios, the fragment intensity ratios, the associated collision energy, and the chromatographic elution time to be optionally used in time-constrained MRM analyses. We will add isotopically labeled internal peptide standards (with known concentrations determined by amino acid analysis) to facilitate absolute quantitation of selected peptides. Assays will be performed on a triple quadropole mass spectrometer at least in duplicate.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make changes and modifications of the invention to adapt it to various usage and conditions and to utilize the present invention to its fullest extent. The preceding preferred specific embodiments are to be construed as merely illustrative, and not limiting of the scope of the invention in any way whatsoever. The entire disclosure of all applications, patents, and publications (including provisional patent application 61/128,688, filed May 23. 2008) cited above and in the figures are hereby incorporated in their entirety by reference.