RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.

62/356636, filed June 30, 2016, and entitled "Chemical Class Compositions From Collision Cross-Section Fragment Ions", the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present disclosure generally relates to determining chemical class compositions present in a sample using collision cross-section fragment ion values.

BACKGROUND

[0003] The structural elucidation of compounds in complex samples (e.g., bio fluids, tissues from plants and animals, foods, etc.) remains a significant analytical challenge. For example, while metabolomics aims to comprehensively profile low- molecular weight compounds, current estimates are in the range of 10,000 metabolites in a typical biological sample. Also, most of the metabolites in any given sample are largely unknown. Manual mass spectra data interpretation is often required. However, knowledge of the expected fragmentation patterns for many scaffolds is limited, thus further complicating analysis. Yet, the need still remains to rapidly identify co- regulated chemical classes for drug discovery and development, metabolomics, lipidomics, and biomarker discovery.

SUMMARY

[0004] The present disclosure provides high-throughput and robust methods of using information obtained from collision cross-section (CCS) of fragment ions to determine the chemical class composition of a sample.

[0005] Also provided are methods for calculating composite values of precursor and product ions to increase measurement selectivity.

[0006] In one aspect, the provided methods can be used as an alternative to, or in combination with other analytical methods to identify and analyze a large number of chemical classes from multiple fragmentation spectra of a complex sample. BRIEF DESCRIPTION OF THE FIGURES

[0007] FIG. 1 depicts a chromatogram of liquid chromatography - ion mobility mass spectrometry (MS) analysis of brain extracts.

[0008] FIG. 2 illustrates the direct analysis (infusion) of human brain extracts using an ion mobility MS instrument.

[0009] FIG. 3 depicts extracted ion chromatograms for fatty acyl groups obtained after data independent fragmentation of complex lipid extracts analyzed using an Ultra

High-Performance Liquid Chromatography method described herein.

[0010] FIG. 4 depicts a comparison of wild type (WT) versus transgenic (TG) lipid extracted for the fatty acyl EPA after a UHPLC-ion mobility MS analysis described herein.

[0011] FIG. 5 illustrates a hierarchical cluster analysis of specific fragment CCS values extracted from plasma samples of control versus radiation treated mice.

[0012] FIG. 6 illustrates a hierarchical cluster analysis of untargeted CCS values from plasma samples of control versus radiation treated mice.

[0013] FIG. 7 illustrates a correlation molecular network analysis of

precursor/fragment CCS, RT values extracted from human brain samples of healthy controls versus subjects with Alzheimer's disease.

[0014] FIG. 8 depicts mirror CCS fingerprint matching for a lipid after

fragmentation induces either in- source or in a collision cell.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0015] In one embodiment, provided are methods of using information obtained from collision-cross section (CCS) of fragment ion values to determine at least one chemical class composition of a sample, the method comprising: a) fragmenting the sample to generate a series of product and precursor ions; separating the product ions using ion- mobility; b) obtaining CCS values of the product ions; and c) matching or correlating the CCS values of the product ions to pre-determined values.

[0016] In one aspect, the experimentally derived ion properties obtained during steps a) and b), such as the m/z ratio, intensity distribution, elution positon, and charge of the parent ions as well as the m/z ratio, intensity distribution, elution positon, charge, and CCS of the precursor ions, may be used in connection with the CCS values of the product ions to determine at least one chemical class composition of a sample. Thus, in one aspect, the methods herein further comprise obtaining at least one ion property selected from m/z ratio, elution position, intensity distribution, charge, and CCS value of the precursor ions; obtaining at least one ion property selected from m/z ratio, elution position, intensity distribution, and charge of the product ions; correlating the at least one ion property of the precursor ions with the corresponding at least one ion property of the parent ions; and matching or correlating the at least one ion property of both the precursor and parent ions to pre-determined values, of the product ions to pre-determined values.

[0017] "Chemical class" or "chemical class composition" refers to the general class to which compounds are grouped based upon their relative structure or function. These classes are readily apparent to one of skill in the art and include, but are not limited to, lipids (e.g., fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides), polyols (e.g., sugar alcohol), polyethers (e.g., paraformaldehyde, polyethylene glycol, polypropylene glycol, and polytetramethylene glycol), polyesters (e.g., polyethylene terephthalate), vitamins (e.g., Vitamin A, Vitamin B l, Vitamin B2, Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B7, Vitamin 9, Vitamin 12, Vitamin C, Vitamin D, Vitamin E, and Vitamin K), antioxidants (e.g., uric acid, glutathione, and melatonin), amino acids (e.g., natural and non-natural amino acids), nucleotides (e.g., purine and pyrimidine nucleotides), hormones (e.g., eicosanoids, steroids, amines, peptides, and proteins), and carbohydrates (e.g., sugars, starch, cellulose, monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The methods described herein are useful in identifying the chemical class or chemical classes present in a sample. It should be understood that the present methods are not limited to only identifying the chemical class composition of a sample, but are also useful to identify the individual compounds failing within the chemical class. For example, in one aspect, the present methods are useful in identifying a chemical class composition (e.g., a lipid). In another aspect, the present methods are useful in identifying the type of lipid present in the sample (e.g., phosphatidylethanolamine such as vinyl ether and acyl versions). It should also be understood that the samples in the present method may contain numerous chemical classes. Thus, the sample herein can comprise two or more chemical class such as e.g., any one of the chemical classes mentioned above.

[0018] The present disclosure includes methods for calculating the collision cross section (CCS) of fragment ions. As used herein, collision cross section (CCS) represents the effective area for the interaction between an individual ion and the neutral gas through which it travels. CCS, is a physicochemical property, and is related to chemical structure and three-dimensional conformation. If certain instances, the experimental CCS of each detected lipid ion can be searched against CCS databases, where available (i.e., compared with a standard or pre-determined value). Methods for calculating CCS can be found in e.g., Paglia et al., Ion Mobility-Derived Collision Cross Section As an Additional Measure for Lipid Fingerprinting and Identification; Anal Chem. 2015 Jan 20;87(2): 1137-44; doi: 10.1021/ac503715v and Paglia et al. Ion Mobility Derived Collision Cross Sections to Support Metabolomics Applications; Anal Chem; 2014 Apr 15;86(8):3985-93; doi: 10.1021/ac500405x. Epub 2014 Mar 28.

[0019] The present disclosure also includes methods of separating components based on charge state using ion mobility. Ion mobility is a gas-phase electrophoretic technique that enables the separation of gas-phase lipid ions within a chamber pressurized with a buffer gas (e.g., nitrogen). The time required for lipid ions to cross the ion-mobility separation cell-the drift time-depends on the collision frequency between the ions and the buffer gas. Thus, drift times are directly related to the shape, size, and charge of the desired ions as well as to the nature of the buffer gas. Methods of performing ion mobility can be found in e.g., Paglia et al., Ion Mobility-Derived Collision Cross Section As an Additional Measure for Lipid Fingerprinting and Identification; Anal Chem. 2015 Jan 20;87(2): 1137-44; doi: 10.1021/ac503715v and Paglia et al. Ion Mobility Derived Collision Cross Sections to Support Metabolomics Applications; Anal Chem; 2014 Apr 15;86(8):3985-93; doi: 10.1021/ac500405x. Epub 2014 Mar 28.

[0020] In one embodiment, the ion mobility comprises the step of performing drift-tube ion mobility spectrometry, travelling-wave ion mobility spectrometry, or differential mobility spectrometry.

[0021] In one embodiment, the ion mobility-mass spectrometry is operated in a data independent acquisition mode or data-dependent acquisition, or a combination thereof, wherein CCS of the precursor ions are associated with CCS of product ions in the sample.

[0022] In one embodiment, the ion mobility-mass spectrometry is operated in a data independent acquisition mode or data-dependent acquisition, or a combination thereof, wherein CCS of the precursor ions are associated with CCS of product ions in the sample; and the method further comprises separating the precursor ions by ion mobility spectrometry, fragmenting the precursor ions, and obtaining the values of the precursor ions; and calculating a hash mass comprising a composite value consisting of the precursor ion value m/z multiplied by the precursor ion collision cross-section area squared (CCSA ) multiplied by the product ion m/z multiplied by the product ion CCSA to determine the association between CCS precursor ions and CCS product ions in the sample.

[0023] In one embodiment, fragmentation of the sample is performed in source.

[0024] Fragmentation of the sample can be induced by means common to one of skill in the art. Such methods include e.g., tandem mass spectrometry (MS/MS), including collision induced dissociation (CID), electron transfer dissociation (ETD), electron impact, ultraviolet photoionization, ozonolysis, or similar fragmentation tools.

[0025] In one embodiment, the ionization method used in the present methods is selected from electrospray ionization (ESI), matrix assisted laser desorption ionization (MALDI) or ambient ionization tools, including laser ablation electrospray ionization (LAESI), desorption electrospray ionization (DESI), and rapid evaporative ionization mass spectrometry (REIMS).

[0026] In one embodiment, the methods herein further comprise separating the chemical classes prior to the separation of ions. Alternatively, the methods herein further comprise separating the chemical classes using chromatography prior to the separation of ions. In another alternative, the methods herein further comprise separating the chemical classes prior to the separation of ions using ion mobility. Such methods include e.g.., liquid chromatography, supercritical fluid chromatography or gas chromatography.

[0027] The samples analyzed by the present methods include sample which are capable of being ionized. In one aspect, the sample is a complex sample, meaning the sample contains multiple components (e.g., one or more compounds). Typically, the sample is biological in nature. In one aspect, the sample herein are biofluids, tissues, sections, or cell cultures.

[0028] The present methods can also be used as means for determining cross- section fragment ion values of a sample and then storing or using those values as a standard for future reference. Thus, in one aspect, the present methods provide the use of drift-time ion mobility spectrometry, travelling-wave ion mobility spectrometry, or differential mobility spectrometry for determining cross-section fragment ion values of a sample, the method comprising: fragmenting the sample to generate a series of product ions; separating the product ions using ion-mobility; obtaining collision cross- section values; and collecting the collision cross-section values.

[0029] The subject technology is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, are incorporated herein by reference.

EXEMPLIFICATION

[0030] Ion Mobility MS Analysis of Brain Extracts

[0031] FIG. 1 shows an analysis of brain extracts using ion mobility mass spectrometry (MS) based on the present methods. The sample (i.e., brain extract) was analyzed using an UHPLC coupled with Synapt-G2Si HDMS in data independent acquisition mode as previously reported. See Paglia et al., Ion Mobility- Derived Collision Cross Section As an Additional Measure for Lipid Fingerprinting and Identification; Anal Chem. 2015 Jan 20;87(2): 1137-44; doi: 10.1021/ac503715v and Paglia et al. Ion Mobility Derived Collision Cross Sections to Support Metabolomics Applications; Anal Chem; 2014 Apr 15;86(8):3985-93; doi: 10.1021/ac500405x. Epub 2014 Mar 28. Alternating high and low energy in the collision cell located before the ion mobility separation cell allowed us to acquire information CCS for both precursors and fragments ions.

[0032] FIG. 2 shows a direct analysis (infusion) of human brain extracts using the present methods. Precursor and fragment ions can be separated using CCS versus m/z plots. Similar fragments cluster together in the CCS-m/z space providing information on their chemical structure.

[0033] As shown in FIG. 3 fragment information can be either extracted using CCS or mass values, or a combination of the above. Typical brain

phosphatidylethanolamine (PE) species are fragmented before ion mobility separation. In negative ionization mode, PEs loose fatty acyl units, whose CCS values can be used for identification. CCS values for common lipid species (e.g., fatty acyl groups) are listed in Table 1. Table 1.

Table 1 - Continued

[0034] FIG. 4 shows a comparison of wild type (WT) versus transgenic (TG) lipid extracted for the specific CCS of the fatty acyl EPA after ion mobility MS analysis using a UHPLC instrument coupled with a Mass Spectrometer (Synapt-G2Si HDMS available from Waters Corporation, Milford MA) in data independent acquisition mode as previously reported. Briefly, fragment information for EPA can be either be extracted using CCS or mass values, or a combination of the above. CCS values for the EPA moiety is listed in Table 1.

[0035] As shown by FIG. 5, specific fragment CCS values of metabolites extracted from plasma samples can be used as biosignature to differentiate between control versus radiation treated mice using a hierarchical cluster analysis of the CCS features that are most altered after radiation exposure. In FIG. 5, no or a small change is represented by a 0 value, illustrated as black in the figure. In FIG. 5 a change in relative abundance (control versus radiated) of matched product ions (same CCS values same m/z) is illustrated by both the color and intensity of the color

(gray=control, white=radiated) for each pixel. It is noted that the top left and bottom right quadrants of FIG. 5 appear to indicate greater change in the control direction, whereas the bottom left and top right quadrants appear to indicate greater change in the radiation direction. Samples were analyzed with a UHPLC instrument coupled with a Mass Spectrometer (Synapt-G2Si HDMS available from Waters Corporation, Milford MA) using a modification of a previously reported method [see Paglia et al., Anal Chem. 2015 Jan 20;87(2): 1137-44. Briefly, in order to generate fragments, the cone (from 1 to 4) and capillary voltages (from 5 to 50 V) were increased and/or the collision energy was ramped in the trap cell (from 5 to 40 eV), which is located before the ion mobility cell, calculating CCS values for all the fragments. Alternating low (5eV) and high collision energy (ramping 20-40 eV) allowed data independent acquisition of CCS values for both precursors and fragments ions.

[0036] UHPLC conditions were as follows: Mobile phase A: 10 mM ammonium formate with 0.1% formic acid in 40:60 (v/v) acetonitrile/water; Mobile phase B: 10 mM ammonium formate with 0.1% formic acid in 10:90 (v/v;.

acetonitrile/isopropanol; Column temperature at 55°C, the flow rate at 0.4 mL/min, the injection volume at 5 μί, and the autosampler temperature at 10°C. Initial conditions started with 40% B and immediately a linear gradient from 40% to 43% B in 2 min. In the following 0.1 min, the percentage of mobile phase B was increased to 50%. Over the next 9.9 minutes, the gradient was further ramped to 54% B, and the amount of mobile phase B was increased to 70% in 0.1 min. In the final part of the gradient, the % B was increased to 99% in 5.9 min. The eluent composition returned to the initial conditions in 0.1 min, and the column was equilibrated, at the initial conditions, for 1.9 min before the next injection. The total run time was 20.0 min.

[0037] Alternatively, the CCS and/or the m/z of precursor ions can be associated with CCS of product ions. Product ions are generated by disassociation of a parent precursor, each; precursor or product ion with its own m/z. Provided two channels of data are collected; one where the precursor ions are separated by IMS then disassociated and a second where precursor ions are disassociated and their corresponding product ions are separated by IMS, a highly specific "hash mass" can be generated. A hash mass is a composite value consisting of a precursor m/z * its CCSA2 * the product ion m/z * its CCSA2. Any combination or mathematical manipulation of the four attributes can be utilized to increase the specificity of a targeted identification. Often in a given MS spectrum two different precursor ions of the same m/z (within the resolving power of the instrument) but different CCSA2 values will be present, similarly two different precursor ions of the same

hydrophobicity but different m/z will produce a fragment ion of similar m/z (within the resolving power of the instrument) fragment ion but different CCSA2. The use of the hash tag can significant increase the selectivity of the identification. FIG. 6 shows a hierarchical cluster analysis of untargeted CCS values from plasma samples of control versus radiation treated mice. Precursor or fragment CCS or a combination of the two can be used. Samples were analyzed with a UHPLC instrument coupled with a Mass Spectrometer (Synapt-G2Si HDMS available from Waters Corporation, Milford MA) as reported above. In this figure, the color key indicates the relative change for control versus radiation treated samples. The absence of change or small change in signal is represented by a value of 0 (and appears as black in the image). A change in relative abundance (control versus radiated) of matched hash masses (CCS times m/z) is illustrated by both the color and intensity of the color (grey=control,

white=radiated) for each pixel. The scale of -1 to 1 represents not an absolute change in abundance, but a scale the brightest color the values (1, -1) being the extremes.

[0038] FIG. 7 shows a correlation molecular network analysis of

precursor/fragment CCS, RT values extracted from human brain samples of healthy control subjects versus subjects with Alzheimer's disease (n=20 per group). This analysis highlights the power of this approach to identify networks of coregulated chemical classed based on the fragments' CCS values. Samples were analyzed with a UHPLC instrument coupled with a Mass Spectrometer (Synapt-G2Si HDMS available from Waters Corporation, Milford MA). The UHPLC-MS acquisition was performed using a modification of a previously reported method (see Anal Chem; 2014 Apr 15;86(8):3985-93). Briefly, in order to generate fragments the cone (from 1 to 4) and capillary voltages (from 5 to 50 V) were increased and/or the collision energy was ramped in the trap cell (from 5 to 40 eV), which is located before the ion mobility cell, calculating CCS values for all the fragments. Alternating low (5eV) and high collision energy (ramping 20-40 eV) allowed data independent acquisition of CCS values for both precursors and fragments ions.

[0039] UHPLC Conditions. Mobile phase A: acetonitrile with 0.1% formic acid in acetonitrile/water 40:60 (vol/vol). Mobile phase B: water with 0.1% formic acid in acetonitrile/isopropanol 10:90 (vol/vol). Column 2.1 x 150 mm)ACQUITY amide column, particle size 1.7 μιη. Column temperature at 45°C, the flow rate at 0.4 mL/min, the injection volume at 5 μί, and the autosampler temperature at 4°C.

Gradient conditions are as follows:

[0040] As shown in FIG. 8 mirror CCS fingerprint matching for a lipid after fragmentation induced either in-source or in a collision cell was performed. This generated an array of product ions each associated with a CCS value. Pure chemicals can be used to build a database of fragments' CCS values which can then be matched against complex samples. Samples were analyzed with a UHPLC instrument coupled with a Mass Spectrometer (Synapt-G2Si HDMS available from Waters Corporation, Milford MA)

[0041] Computationally predicted CCSs can also be performed. In cases where mass measurements are not able to differentiate between potential candidate metabolites obtained from a database search, a comparison between the experimental and theoretical CCS values can support the identification process. Table 2 shows CCS of fragments as measured by experimental condition or computationally predicted.

Table 2.

[0042] Table 3 shows representative fragments for lipids species of

phosphatidylcholines (PC), corresponding to lyso-phosphatidylcholines (LPC). CCS values for LPC species are listed in Table 1.

Table 3.

[0043] Table 4 shows representative fragments for lipids species of

phosphatidylethanolamines (PE), corresponding to lyso-phosphatidylethanolamines (LPE). CCS values for LPE species are listed in Table 1.

Table 4.