WO2013173642A1 - High-throughput lipidomics - Google Patents

Abstract

Provided are methods identifying a plurality of lipids in a sample that entail subjecting a first portion of the sample to mass spectrometry to obtain a full mass spectrum comprising a plurality of detected full mass values, subjecting a second portion of the sample to mass spectrometry to obtain an all-ion-fragmentation (AIF) mass spectrum comprising a plurality of detected ion fragment mass values, identifying, for each detected full mass value, one or more candidate lipids having a matched theoretical mass value, from a database comprising a plurality of lipids, a list of ion fragments for each of the lipids, and a theoretical mass value for each of the lipids and each of the ion fragments, retrieving from the database, for each of the candidate lipids, the corresponding ion fragments and theoretical mass values of the fragments, and determining that if the plurality of detected ion fragment mass values include values that match the theoretical mass values of the corresponding ion fragments of one or more of the matched lipids, then the one or more of the matched lipids are present in the sample, thereby identifying a plurality of lipids in the sample.

Description

HIGH-THROUGHPUT LIPIDOMICS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional

Application No. 61/648,823, filed May 18, 2012, the contents of which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Lipidomics, metabolomics of lipids, is a research field that studies cellular lipidomes on a large scale and at the intact-molecule level. Precise identification and quantification of individual cellular lipid species and understanding their cellular function at the systems biology level are the primary tasks of the field at its current stage. Advances in mass spectrometry development of the soft ionization techniques of electrospray ionization (ESI) and matrix- assisted laser desorption/ionization (MALDI) greatly facilitate the progress of modern lipidomics.

[0003] There exist two major mass spectrometry (MS)-based platforms in current lipidomics practice (i.e., the direct infusion approach (shotgun lipidomics) and chromatographic separation coupled MS (LC-MS)). In instrumentation, low mass accuracy/resolution mass spectrometers (e.g., quadrupole-based instruments) are the major working horses in both platforms. Examples include multidimensional MS-based shotgun lipidomics (MDMS-SL), tandem MS-based shotgun lipidomics, and selected ion monitoring or selected (multiple) reaction monitoring (SRM/MRM)- based LC-MS approaches. These platforms generally fall into a time frame of an approximately hour to hours for global analysis of cellular lipidomes.

[0004] As the mass resolution of bench top instruments including quadrupole-time of flight type and Orbitrap advanced to a scale of 40,000 or higher around m/z 500, shotgun lipidomics platforms have progressed to the high throughput fashion in a time scale of minutes to close to an hour. All these platforms have greatly facilitated lipidomics research to allow our understanding of the roles of lipids in biological systems in health and their alterations during disease processes.

[0005] Nowadays, the mass resolution has achieved to a benchmark of > 140,000 at m/z 200 with a mass accuracy of < 0.1 ppm on the bench top instrument (e.g., Thermo Scientific Q- Exactive mass spectrometer). How to take advantage of this progress to further advance our analytical technology to a new level of throughput for lipidomics research would indeed be both a chance and a challenge.

SUMMARY

[0006] This disclosure presents a platform for the analysis of cellular lipidomes in an ultra high-throughput fashion (e.g., in a time frame of seconds). By combining the accurate masses of individual lipid species determined in a full mass spectrum with those specific fragmentation patterns of neutral losses present in an all-ion-fragmentation (AIF) mass spectrum of lipid extract, this platform can identify the structures of individual species of an entire class (including subclasses) of interest including the identities of aliphatic chains (except the location of double bond(s)), regiospecificity of each species if present, and the composition of isomeric species. This technology is achieved with the accompanying development of a new method for real-time data processing. As shown in the experimental examples, this platform allowed the analysis of nearly 15 lipid classes and hundreds of individual molecular species directly from lipid extracts of biological samples.

[0007] Provided, in one embodiment, is a method for identifying a plurality of lipids, and/or quantitating the lipids, in a sample, comprising subjecting a first portion of the sample to mass spectrometry to obtain a full mass spectrum comprising a plurality of detected full mass values; subjecting a second portion of the sample to mass spectrometry to obtain an all-ion- fragmentation (AIF) mass spectrum comprising a plurality of detected ion fragment mass values; identifying, for each detected full mass value, one or more candidate lipids having a matched theoretical mass value, from a database comprising a plurality of lipids, a list of ion fragments for each of the lipids, and a theoretical mass value for each of the lipids and each of the ion fragments; retrieving from the database, for each of the candidate lipids, the corresponding ion fragments and theoretical mass values of the fragments; and determining that if the plurality of detected ion fragment mass values include values that match the theoretical mass values of the corresponding ion fragments of one or more of the matched lipids, then the one or more of the matched lipids are present in the sample, thereby identifying a plurality of lipids in the sample.

[0008] In some aspects, the corresponding ion fragments of each candidate lipid comprise at least two neutral loss (NL) fragments of the candidate lipid. In some aspects, the AIF mass spectrum comprises results from at least two scans at different collision energies. [0009] In some aspects, the AIF mass spectrum comprises a positive-ion mode mass spectrum. In some aspects, the AIF mass spectrum comprises a negative-ion mode mass spectrum.

[0010] In some aspects, the full mass spectrum covers at least a range of 500-800 mass charge (m/z) ratios. In some aspects, the full mass spectrum covers at least a range of 350-1000 m/z ratios.

[0011] In some aspects, the AIF mass spectrum covers at least a range of 200-800 m/z ratios. In some aspects, the AIF mass spectrum covers at least a range of 50-1000 m/z ratios.

[0012] In some aspects, the method further comprises derivatizing the sample prior to mass spectrometry. In some aspects, the method further comprises enriching the content of lipids in the sample with organic solvent extraction or chromatography.

[0013] In some aspects, the sample is a biological sample. In some aspects, the biological sample comprises a body fluid.

[0014] Also provided, in one embodiment, is a method for identifying a plurality of lipids in a sample, comprising receiving, at a computer, a full mass spectrum comprising a plurality of detected full mass values, generated from the sample; receiving an all-ion-fragmentation (AIF) mass spectrum comprising a plurality of detected ion fragment mass values, generated from the sample; identifying, for each detected full mass value, one or more candidate lipids having a matched theoretical mass value, from a database comprising a plurality of lipids, a list of ion fragments for each of the lipids, and a theoretical mass value for each of the lipids and each of the ion fragments; retrieving from the database, for each of the candidate lipids, the

corresponding ion fragments and theoretical mass values of the fragments; and determining that if the plurality of detected ion fragment mass values include values that match the theoretical mass values of the corresponding ion fragments of one or more of the matched lipids, then the one or more of the matched lipids are present in the sample, thereby identifying a plurality of lipids in the sample.

[0015] The present disclosure also provides, in one embodiment, a method for identifying the composition of a mass spectrum peak, comprising subjecting a sample comprising a plurality of lipids to mass spectrometry to obtain a mass spectrum comprising at least a mass spectrum peak; identifying a first lipid that is suspected to be present in the peak; generating a list of lipids that have identical number of carbons as the first lipid but have more or fewer double bonds than, or have the same number of double bonds as, the first lipid; determining whether the peak is an aggregate of two or more peaks formed by lipids from the list.

13

[0016] In some aspects, the method further comprises comparing the peak to the C

13

isotopologues of the lipids in the list to determine whether any of the C isotopologues is present in the peak.

[0017] Also provided, in some embodiments, is a method for identifying the composition of a peak in mass spectrum, comprising receiving, at a computer, a mass spectrum generated from a sample comprising a plurality of lipids, wherein the mass spectrum comprises at least a mass spectrum peak; identifying a first lipid that is suspected to be present in the peak; generating a list of lipids that have identical number of carbons as the first lipid but have more or fewer double bonds than, or have the same number of double bonds as, the first lipid; determining whether the peak is an aggregate of two or more peaks formed by lipids from the list. In some aspects, the

13

method further comprises comparing the peak to the C isotopologues of the lipids in the list to

13

determine whether any of the C isotopologues is present in the peak.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1A illustrates the concept of one embodiment of the present technology, in which the upper panel shows a full mass spectrum and the one below is an all-ion-fragmentation (AIF) mass spectrum. Possible neutral loss (NL) fragments from candidate lipids detected from the full mass spectrum are searched in the AIF mass spectrum.

[0019] FIG. IB illustrates that the AIF mass spectrum can pool data from three scans with different energies.

[0020] FIG. 2 is a schematic illustration of the shotgun lipidomics platform assisted with accurate masses of the neutral loss fragments resulted from a molecular ion. The platform is comprised of four components including (1) sample preparation in which lipid extracts are derivatized with CD₃I to convert ethanolamine-containing lipid classes into choline-containing lipids so that these species can possess a specific NL fragmentation pattern essentially identical to choline glycerophospho lipid (PC) species; (2) selective ionization of the lipid solution in both positive-ion mode in the presence of LiOH and negative-ion mode; (3) MS analysis of the lipid solution in each mode with a full mass and an all-ion- fragmentation (AIF) mass spectrum; and (4) processing and analysis of MS dataset to identify and quantify individual species of lipid classes as listed utilizing an in-house developed algorithm of accurate neutral loss-assisted shotgun lipidomics (ANLA-SL). Lipid classes abbreviated SM, PE, MMPE, DMPE, PS, PI, PG, PA, Cer, LPC, LPE, HexCer, and AC stand for sphingomyelin, ethanolamine glycerophospholipid, N- monomethyl PE, Ν,Ν-dimethyl PE, phosphatidylserine, phosphatidylinositol,

phosphatidylglycerol, phosphatidic acid, ceramide, lysoPC, lysoPE, monohexosylceramide, and acylcarnitine, respectively.

[0021] FIG. 3 shows a schematic workflow of the ANLA-SL platform for identification and quantification of individual lipid species assisted with accurate neutral loss fragments.

[0022] FIG. 4A-B show mass spectrometric analysis of PC species in lipid extracts utilizing an ultra high-resolution/mass accuracy mass spectrometer. A specific NL fragmentation pattern resulted from a lithiated PC species was exemplified in a schematic product-ion mass spectrum (A). Portions of an AIF mass spectrum, each of which includes neutral loss ion fragments corresponding to a candidate lipid as shown (indicated with arrows, as a, b, and c) (B).

[0023] FIG. 5A-B show the product ion mass spectra of lithiated acylcarnitine (A) and lithiated lysosphingomyelin (B).

[0024] FIG. 6A-B show the double bond overlapping effects that complicate the isotopic patterns of lipid species analyzed by high mass resolution shotgun lipidomics. A shows the relative mass defects of different elements to one or two C mass. C stands for C. D and N

13

represent deuterium and nitrogen- 15 relative to one C atom. O, S, and CI denote oxygen- 18,

13

sulfer-34, and chlorine-37 relative to two C atoms, respectively. DB represents the mass defects

13

of one less double bond (i.e., addition of two protons) relative to two C atoms. B indicates the differential mass shifts at ppm of isotopologues containing these isotopes relative to ¹³C atom(s) at m/z 700 where the majority of lipid ions are detected by ESI-MS.

[0025] FIG. 7A-F present the simulation results of the mass spectra displaying the M+2 13 C isotopologue and the ion (L) which differs one double bond from the species M with different instrumental mass resolution. An equal intensity ratio of the M+2 isotopologue and the L ion from the classes of both PC and PI was used. The mass spectra of these ions with instrumental mass resolution of 600K (Panel A and D), 150K (Panels B and E), and 75K (Panels C and F) were simulated as described in Example 2. [0026] FIG. 8 shows the determination of the mass shift with standard phosphatidylcholine species resulted from the double bond overlapping effect in a molar ratio dependent manner. Mixtures of standard 16:0-18:0 and 16:0-18: 1 PC species were prepared and analyzed by using a Q-Exactive mass spectrometer with mass resolution of 75k around m/z 700 as described in the subsection of "Materials and Methods". Mass spectra of the mixtures comprised of 1 : 10, 1 : 1, and

13

10: 1 ratios of 16:0-18: 1 PC M+2 C isotopologue vs. 16:0-18:0 PC ion were displayed in Panels A to C, respectively, indicating the overlapping peak shape and the mass shift due to the overlapping. Panel D demonstrated the determined locations of ions corresponding to 16:0-18: 1 (solid cycles) and 16:0-18:0 (open cycles) PC species relative to their theoretical values of 766.5932605 and 768.60891056 Da, respectively. Lock mass of 766.5932605 Da (i.e., 16:0-18: 1 PC species) was used during mass spectral acquisition. The variation of the mass shift corresponding to 16:0-18:0 PC was observed as changed in the total concentration of the PC mixtures.

DETAILED DESCRIPTION

Definitions

[0027] As used herein, unless otherwise stated, the singular forms "a," "an," and "the" include plural reference. Thus, for example, a reference to "a lipid" includes a plurality of lipid molecules.

[0028] As used herein, the term "purification" or "purifying" does not refer to removing all materials from the sample other than the analyte(s) of interest. Instead, purification refers to a procedure that enriches the amount of one or more analytes of interest relative to other components in the sample that may interfere with detection of the analyte of interest.

Purification of the sample by various means may allow relative reduction of one or more interfering substances, e.g. , one or more substances that may or may not interfere with the detection of selected lipid parent or daughter ions by mass spectrometry. Relative reduction as this term is used does not require that any substance, present with the analyte of interest in the material to be purified, is entirely removed by purification.

[0029] As used herein, the term "test sample" refers to any sample that may contain lipids. As used herein, the term "body fluid" means any fluid that can be isolated from the body of an individual. For example, "body fluid" may include blood, plasma, serum, bile, saliva, urine, tears, perspiration, and the like. [0030] As used herein, the term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

[0031] As used herein, the term "liquid chromatography" or "LC" means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s). Examples of "liquid chromatography" include reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UPLC), and high turbulence liquid chromatography (HTLC).

[0032] As used herein, the term "high performance liquid chromatography" or "HPLC" refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase on a support matrix, typically a densely packed column. As used herein, the term "ultra high performance liquid chromatography" or "UPLC" or "UHPLC" (sometimes known as "ultra high pressure liquid chromatography") refers to HPLC which is conducted at higher pressures than traditional HPLC techniques (ca. > 5000 psi) and optionally with column packing materials with smaller particle sizes (ca. < 5 μηι).

[0033] As used herein, the term "high turbulence liquid chromatography" or "HTLC" refers to a form of chromatography that utilizes turbulent flow of the material being assayed through the column packing as the basis for performing the separation. HTLC has been applied in the preparation of samples containing two unnamed drugs prior to analysis by mass spectrometry. See, e.g., Zimmer et ah, J. Chromatogr. A 854: 23-35 (1999); see also, U.S. Patents No.

5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further explain HTLC. Persons of ordinary skill in the art understand "turbulent flow". When fluid flows slowly and smoothly, the flow is called "laminar flow". For example, fluid moving through an HPLC column at low flow rates is laminar. In laminar flow the motion of the particles of fluid is orderly with particles moving generally in straight lines. At faster velocities, the inertia of the water overcomes fluid frictional forces and turbulent flow results. Fluid not in contact with the irregular boundary "outruns" that which is slowed by friction or deflected by an uneven surface. When a fluid is flowing turbulently, it flows in eddies and whirls (or vortices), with more "drag" than when the flow is laminar.

[0034] As used herein, the term "gas chromatography" or "GC" refers to chromatography in which the sample mixture is vaporized and injected into a stream of carrier gas (as nitrogen or helium) moving through a column containing a stationary phase composed of a liquid or a particulate solid and is separated into its component compounds according to the affinity of the compounds for the stationary phase.

[0035] As used herein, the term "on-line" or "inline", for example as used in "on-line automated fashion" or "on-line extraction" refers to a procedure performed without the need for operator intervention. In contrast, the term "off-line" as used herein refers to a procedure requiring manual intervention of an operator. Thus, if samples are subjected to precipitation, and the supernatants are then manually loaded into an autosampler, the precipitation and loading steps are off-line from the subsequent steps. In various embodiments of the methods, one or more steps may be performed in an on-line automated fashion.

[0036] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique to identify compounds by their mass. MS refers to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m/z". MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio. The compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields or other combination(s), the ions follow a path in space or time domain that is dependent upon mass ("m") and charge ("z").

[0037] As used herein, the term operating in "negative ion mode" refers to those mass spectrometry methods where negative ions are generated and detected. The term operating in "positive ion mode" as used herein, refers to those mass spectrometry methods where positive ions are generated and detected.

[0038] As used herein, the term "ionization" or "ionizing" refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge lost with one or more electron units.

[0039] As used herein, the term "electron ionization" or "EI" refers to methods in which an analyte of interest in a gaseous or vapor phase interacts with a flow of electrons. Impact of the electrons with the analyte produces analyte ions, which may then be subjected to a mass spectrometry technique.

[0040] As used herein, the term "chemical ionization" or "CI" refers to methods in which a reagent gas (e.g. ammonia) is subjected to electron impact, and analyte ions are formed by the interaction of reagent gas ions and analyte molecules.

[0041] As used herein, the term "matrix-assisted laser desorption ionization" or "MALDI" refers to methods in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy- absorbing matrix, which facilitates desorption of analyte molecules.

[0042] As used herein, the term "surface enhanced laser desorption ionization" or "SELDI" refers to another method in which a non- volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo- ionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is typically bound to a surface that preferentially retains one or more analytes of interest. As in MALDI, this process may also employ an energy-absorbing material to facilitate ionization.

[0043] As used herein, the term "electrospray ionization" or "ESI," refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Solution reaching the end of the tube is vaporized

(nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows through an evaporation chamber. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

[0044] The term "about" as used herein in reference to quantitative measurements not including the measurement of the mass of an ion, refers to the indicated value plus or minus 10%. Mass spectrometry instruments can vary slightly in determining the mass of a given analyte. The term "about" in the context of the mass of an ion or the mass/charge ratio of an ion refers to +/- 0.50 atomic mass unit.

Accurate Neutral Loss-Assisted Shotgun Lipidomics (ANLA-SL)

[0045] With recent advances in bench-top ultra high resolution instrumentation, one of the important developments is to strategically maximize the utilization of high resolution mass spectrometry for reliable and ultra high-throughput shotgun lipidomics. The presently described lipidomics platform is based on high-resolution mass spectrometry (MS) and accurate neutral loss (NL) fragmentation. Throughout the disclosure, this platform is referred to as accurate neutral loss-assisted shotgun lipidomics (ANLA-SL).

[0046] FIG. 1-3 illustrate one embodiment of the lipidomics procedure of the present disclosure. For each sample, at least a full mass MS scan and an all-ion-fragment (AIF) MS scan are carried out (see, e.g., FIG. 2). The upper panel of FIG. 1A shows an example full mass spectrum. In the full mass spectrum, if an ion(s) represented by a detected peak matches with the true mass value of a species of a lipid class possessing the charge (e.g., with an accuracy of approximately 0.5 ppm after correction for overlapping effect(s) by accurate mass searching (see above)), the system can identify the ion(s) as a species (a "candidate lipid") of the class. As FIG. IB illustrates, each full mass spectrum and/or all-ion-fragment mass spectrum which can include pooled data from multiple scans carried out at different collision energy levels.

[0047] The structure(s) of the candidate lipid(s) is still not definitively identified at this moment as there are multiple possibilities. The structure(s), however, can be identified by searching for the existence of its (or their) unique neutral loss (NL) fragmentation pattern(s) present in the corresponding AIF mass spectrum. It is recognized that NL scanning is molecular species specific. Specifically, if this mass-matched candidate lipid represents a species of the class, the specific NL fragmentation pattern yielding from this species should exist in the AIF mass spectrum. If such a pattern does not exist, then this candidate lipid does not exist in the sample.

[0048] If the accurate mass of this candidate lipid matches with those of multiple isomeric species of a class, then all the NL fragmentation patterns of those isomeric species should exist in the AIF mass spectrum. Further, the regiospecificity of the paired fatty acyl chains in an identified diacylglycerol (DAG)-derived species is determined utilizing the ratio of ion intensities of the fragments corresponding to the NL of the fatty acids (FAs). [0049] Thus, one embodiment of the disclosure provides a method for identifying and/or quantitating a plurality of lipids in a sample. The method, in one embodiments, entails subjecting a first portion of the sample to mass spectrometry to obtain a full mass spectrum comprising a plurality of detected full mass values, and subjecting a second portion of the sample to mass spectrometry to obtain an all-ion- fragmentation (AIF) mass spectrum comprising a plurality of detected ion fragment mass values. In some aspects, the sample is subjected to tandem mass spectrometry.

[0050] For each detected full mass value, the method identifies one or more candidate lipids having a matched theoretical mass value. Such identification can be carried out by looking up a lipidomics database. The database, in some aspects, includes lipids, identification of the lipids and their theoretical mass values. Further, also in the same database or in a separate database, there exist information about ion fragments for each of the lipids and theoretical mass values for these fragments. In some aspects, included are all possible ion fragments for each lipid. In some aspects, the database includes two or more possible neutral loss (NL) fragments for each lipid.

[0051] Candidate lipids identified from the full mass spectrum by mass value-matching can be further checked and verified with information obtained from the AIF mass spectrum. To do that, once a candidate lipid is identified from the full mass spectrum, the system retrieves from the database the corresponding ion fragments for the candidate lipid. Subsequently, the method checks the AIF mass spectrum to determine whether such corresponding ion fragments are present there, by mass value-matching assisted with a neural loss fragmentation pattern.

[0052] As provided, if the AIF mass spectrum does not contain all of the corresponding ion fragments, then it can be concluded that the candidate lipid actually does not exist in the sample. Then, another candidate lipid can be checked or verified, by mass value-matching (after correction for ion overlaps) with the AIF mass spectrum with its corresponding ion fragments. It is possible that more than one candidate lipids are represented by one peak in the full mass spectrum.

[0053] By checking every candidate lipid identified from the full mass spectrum against the AIF mass spectrum, using a list of its possible ion fragments, in particular NL fragments, this method can identify all lipids represented by the full mass spectrum peaks and their specific structures. [0054] The methods of the present disclosure can also quantitate the lipids identified from the mass spectra. What can be determined is whether the full intensity of an ion represents the abundance of the identified species described above, but not from other hidden isobaric component(s) from other lipid classes under the condition of high mass resolution. The method, on one embodiment, calculates an intensity ratio of M+l isotopologue and monoisotopic peak in the full mass spectrum. A ratio that equals to the theoretical value of the identified species can indicate that an isobaric species does not co-exist. A ratio that is less than the theoretical value can indicate the existence of isobaric species and the method uses the intensity of the M+l isotopologue to derive the monoisotopic ion intensity for quantification. A ratio that is greater than the theoretical value can indicate the existence of an isobaric species with the M+l isotopologue. In this case, the method employs the intensity of monoisotopic peak for quantification.

[0055] Further, the mass level of individual identified ion corresponding to a lipid class of interest can be determined by ratiometric comparison of the ion intensity to that of the selected internal standard of the class detected in the full mass spectrum. A wide linear dynamic range for quantification can be present by ratiometric comparison utilizing ion intensities detected in the full mass spectrum under certain conditions. These conditions include (1) at a low lipid concentration (to avoid lipid aggregation); (2) for a polar lipid class (to minimize the effects of differential acyl chain contribution); (3) after ¹³C de-isotoping for monoisotopic peak comparison as well as to resolve any overlap between an m+2 isotopomer of an ion A with an ion containing one double bond less than the ion A; and (4) only utilizing a full mass spectrum to avoid any differential fragmentation of different molecular species of the class. The composition of the isomeric species of an ion (if present) can be estimated from the intensity ratio of fragments corresponding to the NL of either sn-1 or sn-2 FAs.

Lipid Enrichment, Full Mass Spectrum and All Ion Fragment Mass Spectrum

[0056] As provided, each sample is subjected to both a full mass spectrum and an all ion fragment (AIF) mass spectrum. Prior to the mass spectrometry, in some aspects, a sample such as a biological sample, can be processed to enrich the lipid content. The lipids in the sample, in some aspects, can be derivatized to facilitate the analysis.

[0057] For instance, all lipid samples analyzed can be prepared through organic solvent extraction, with or without column separation, and/or gas or liquid chromatography. This procedure can eliminate all or most non-lipid candidates of isomeric species. Then, a lipid extract of a biological sample can be derivatized with deuterated methyl iodide (CD₃I) to convert ethanolamine glycerophospholipid (PE), lyso PE (LPE), N- monomethyl PE (MMPE), and N,N- dimethyl PE (DMPE) to d₉-choline glycerophospholipid (d₉- PC), d₉-LPC, d₆-PC, and d₃-PC, respectively (FIG. 2). It is noted that in addition to the derivatization with CD₃I, other chemical derivatization/reaction could also be used to prepare a sample for the analysis of a particular category of lipid classes (e.g., alkaline methanolysis for the enhanced analysis of sphingolipids if necessary).

[0058] Employing this procedure can ensure the unique NL fragmentation patterns of lithiated PC and LPC species for the analysis of ethanolamine-containing lipid species with the ANLA-SL platform since these unique NL fragmentation patterns allow definitive identification of individual molecular species of cho line-containing lipid classes.

[0059] It is noted that the yield of the chemical reaction does not significantly affect the analysis of these lipid classes due to the presence of an internal standard for each individual lipid class. However, the experimental conditions can be optimized to achieve a nearly complete reaction.

[0060] A sample can be split into two or more aliquots, allowing each aliquot to undergo different mass spectrometry scan. In some aspects, one aliquot is used for mass spectrometry analysis in the positive-ion mode and the other well is for the negative-ion mode. This procedure was designed to achieve a selective ionization of different lipid classes (FIG. 2).

[0061] In each ionization mode, one full mass spectrum and an AIF mass spectrum are acquired. A "full mass spectrum" refers to a mass spectrum, which is either acquired in a single scan or as a combination of multiple scans, that cover all or substantially all lipid classes of interest or in a sample, such as a biological sample. In one aspect, the lipid species are not fragmented. In one aspect, a full mass spectrum ranges from at least about m/z 500 to 800. In one aspect, a full mass spectrum ranges from at least about m/z 500 to 850, or alternatively 500 to 900, 500 to 950, 500 to 1000, 500 to 1050, 500 to 1 100, 500 to 1200, 450 to 800, 450 to 850, 450 to 900, 450 to 1000, 450 to 1050, 450 to 1 100, 450 to 1200, 400 to 800, 400 to 850, 400 to 900, 400 to 950, 400 to 1000, 400 to 1 150, 400 to 1200, 350 to 800, 350 to 850, 350 to 900, 350 to 950, 350 to 1000, 350 to 1050, 350 to 1 100, 350 to 1 150, 350 to 1200, 300 to 800, 300 to 850, 300 to 900, 300 to 950, 300 to 1000, 300 to 1050, 300 to 1 100, 300 to 1 150, 300 to 1200, 250 to 800, 250 to 850, 250 to 900, 250 to 950, 250 to 1000, 250 to 1050, 250 to 1100, 250 to 1150, or 250 to 1200, 250 to 1300, 250 to 1400 or 250 to 1500 (m/z).

[0062] An "all-ion- fragment (AIF)" mass spectrometry refers to a mass spectrometry, which is either acquired in a single scan or as a combination of multiple scans, that covers all or substantially all fragment ions from lipid species of interest or in a sample, which are present in a selected full mass spectrum window. In some aspects, an AIF covers at least one or two or three neutral loss (NL) fragments for each lipid species of interest or in the sample. In one aspect, an AIF mass spectrum ranges from at least about m/z 200 to 800. In one aspect, an AIF mass spectrum ranges from about m/z 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400 to about m/z 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1500.

[0063] As used herein, "substantially all" refers to a proportion of lipid classes in a sample that is at least about 70%, or alternatively at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9% or 99.99% lipid classes.

[0064] As demonstrated in Example 1, a full mass spectrum (ranged from m/z 350 to 1000 which covers majority of the lipid classes of interest) and an AIF mass spectrum ranged from m/z 50 to 1000 were acquired (FIG. 2). For the AIF MS analysis, for instance, the quadrupole was set to pass all ions ranged from m/z 350 to 1000 (called as the Q-window). Five full mass and 10 AIF scans at a speed of 4 Hz were acquired and averaged for each full mass or AIF mass spectrum. Accordingly, all the mass spectra necessary for lipidomics analysis can theoretically be done in a few seconds (e.g., 4 seconds).

[0065] As illustrated in FIG. IB, to obtain a suitable full mass spectrum and/or a suitable AIF mass spectrum, multiple scans at different normalized collision energy (NCE) levels can be carried out and the results are pooled. In one aspect, at least one, or two, or three different collision energy levels are employed.

Mass Search of Candidate Lipid Species

[0066] In one of the steps, once the full mass spectrum and the AIF mass spectrum are obtained, the peaks in the mass spectra are compared to theoretical mass values in a database to determine the identity of the peaks. For instance, when processing the peaks in a full mass spectrum, the method extracts the masses and intensities of each individual ions determined from the full mass spectra. Filtering of the detected masses of ions with theoretical true values of the species of a class of interest in a mass accuracy of 1 ppm yielded a list of the detected molecular species candidates of the class after correction for ion overlaps. Alternatively, the present disclosure provides an improved method considering the ion overlaps for such mass search.

[0067] In shotgun lipidomics, there exists a dogma in the field that if mass accuracy and resolution of a mass spectrometer is high enough, individual molecular species of a lipid class can be accurately searched through comparison with a theoretical lipid database and its peak intensity can be extracted from the mass spectral datasets acquired with such an instrument. Such a database can be readily constructed based on the elementary composition. Therefore, analysis of individual lipid species can be achieved, at least partially, through accurate mass searching from the mass spectra acquired with those instruments.

[0068] To such reasoning, there are a few questions to be answered. For example, what is the minimal mass resolution of an instrument required for such a purpose and if currently commercially-available instruments do not meet the requirement, whether can one develop an approach to achieve such a goal with a relatively lower mass resolution instrument? At present, the mass resolution of the majority of the commercially available mass spectrometers is not high enough to totally resolve the overlaps present in the analysis by shotgun lipidomics. is discovered, however, that the major issue is the partial overlap between the two 13

[0069] It C atom-containing isotopologue of a species M (i.e., M+2 isotopologue) and the ion of a species containing a less double bond than M (assigned here as L) (referred to as "the double bond overlapping effect" hereafter). This issue is not problematic in the case of unit mass resolution mass spectrometry where these ions are completely overlapped each other and the intensity of the overlapped peak is adductive. Therefore, the intensity of each individual ion can be extracted after de-isotoping.

[0070] Example 2 simulated this overlap along with the existence of other isotopic atoms

18 15 34 37

including D, O, N, S, and CI under a variety of experimental conditions and different mass resolution of instruments. It is recognized that this partial overlap alone could cause a mass shift of the species L to the lower mass end up to 13 ppm depending on the molar ratio of species M and L and mass resolution of the instrument used. However, if a mass-searching window of ± 13 ppm was used for accurate mass searching, a substantial number of false positive and nonspecific hits could be included. Therefore, such direct mass searching of a high accurate mass spectral dataset for shotgun lipidomics analysis definitely leads to a complicated outcome. [0071] Based on these findings, this disclosure provides a method for accurate mass searching by exploring one of the major features of lipidomics data that molecular species of a lipid class are present in ion clusters where neighboring masses from different species differ by one or a few double bonds.

[0072] In other words, for a candidate lipid species present in a mass spectrum, the method generates a list of lipids that differ from each other by one or a few double bonds. Then, by comparing such a list to the relative peaks in the mass spectrum, one can more accurately determine the composition of the peaks.

[0073] Thus, in one embodiment, provided is a method for identifying the composition of a mass spectrum peak, comprising subjecting a sample comprising a plurality of lipids to mass spectrometry to obtain a mass spectrum comprising at least a mass spectrum peak; identifying a first lipid that is suspected to be present in the peak; generating a list of lipids that have identical number of carbons as the first lipid but have more or fewer double bonds than, or have the same number of double bonds as, the first lipid; determining whether the peak is an aggregate of two or more peaks formed by lipids from the list. In some aspects, the method further entails comparing

13 13 the peak to the C isotopologues of the lipids in the list to determine whether any of the C isotopologues is present in the peak.

[0074] This approach, for instance, first opens the mass-searching window to 13 ppm to search an entire group of species of a lipid class; these species contain an identical number of carbon atoms. Then accurate mass searching of the plus one ¹³C isotopologue can be used to eliminate

13 13 the potential false positive since the overlaps of the plus one C isotopologue with other C isotopologues are minimal and these overlaps if present are very different from the overlaps

13

between M+2 C isotopologues and L ions. This new mass searching approach is validated through comparing with the species determined by unit-resolution MS (i.e., multi-dimensional MS-based shotgun lipidomics (MDMS-SL)), as shown in Example 2.

Computer Methods and Systems

[0075] The methodology described here can be implemented on a computer system or network. A suitable computer system can include at least a processor and memory; optionally, a computer-readable medium that stores computer code for execution by the processor. Once the code is executed, the computer system carries out the described methodology. [0076] In this regard, a "processor" is an electronic circuit that can execute computer programs. Suitable processors are exemplified by but are not limited to central processing units, microprocessors, graphics processing units, physics processing units, digital signal processors, network processors, front end processors, coprocessors, data processors and audio processors. The term "memory" connotes an electrical device that stores data for retrieval. In one aspect, therefore, a suitable memory is a computer unit that preserves data and assists computation. More generally, suitable methods and devices for providing the requisite network data transmission are known.

[0077] Also contemplated is a non-transitory computer readable medium that includes executable code for carrying out the described methodology. In certain embodiments the medium further contains data or databases needed for such methodology.

EXAMPLES

Example 1

[0078] This example demonstrates a platform for the analysis of cellular lipidomes in an ultra high-throughput fashion (in a time frame of seconds) by using an ultra high-resolution/high mass accuracy mass spectrometer. By combining the accurate masses of individual lipid species determined in a full mass spectrum with those specific fragmentation patterns of accurate neutral losses present in an all-ion- fragmentation (AIF) mass spectrum of lipid extract, this platform can definitively identify the structures of individual species of an entire class (including subclasses) of interest including the identities of aliphatic chains (except the location of double bond(s)), regiospecificity of each species if present, and the composition of isomeric species.

[0079] This technology was achieved with accompanying the development of a new computer program for real-time data processing. This new platform is referred to as "accurate neutral loss- assisted shotgun lipidomics (ANLA-SL)" due to the importance of a specific neutral loss (NL) fragmentation pattern for identification and the requirement of ultra high resolution/high mass accuracy to achieve the extraction of accurate NL masses.

[0080] As shown here, ANLA-SL allows the analysis of nearly 15 lipid classes and hundreds of individual molecular species directly from lipid extracts of biological samples. This example further applied this platform for the analysis of lipids present in human plasma samples. Both the coverage and the mass levels of lipid species in plasma were well comparable with those obtained from other platforms. This technology can be further expanded to many other lipid classes and the platform can be efficiently used for identification and quantitation of hundreds of lipid species from any biological source materials.

[0081] In this example, a Triversa Nanomate device was used for automated infusion of samples; a full mass scan and an all-ion-fragmentation (AIF) MS/MS scan were acquired in a time frame of seconds in both negative and positive modes. MS resolution was set to the highest possible resolution available. Data were processed with an in-house developed software package for fast processing of large numbers of high-resolution datasets.

[0082] This example used lithiated choline glycerophospholipid (PC) species which were selectively ionized and detected in the positive-ion mode. With less than 1 ppm searching window after correction for ion peak overlaps, candidate PC species were matched with those of theoretical values in database. The structures including regiospeficity and fatty acyl chains of these candidate species were further identified with their specific neutral loss fragmentation patterns.

[0083] Custom-designed computer programs were used to screen the AIF spectrum with fragments of PC species with accurate neutral losses. Those identified species were easily quantified after de-isotoping and comparing with PC internal standard using the full mass scan. This example compared the obtained data with those yielded from another lipidomics platform. The two sets of data were fully compatible. The broad dynamic range of the ultra high-resolution instrument (comparing to the full scan data from the triple quadruple instruments) allowed detection of very low abundance PC species.

[0084] This example further extended this strategy for the analysis of over ten lipid classes including PC (diacyl, alkyl-acyl, and plasmalogen species), ethanolamine glycerophospholipids (PE, diacyl, alkyl-acyl, and plasmalogen species), N-methyl PE, Ν,Ν-dimethyl PE,

phosphatidylinositol, lysoPC, lysoPE, phosphatidylglycerol, phosphatidylserine, ceramide, hexosylceramide, sphingomyelin, acylcarnitine. Collectively, in aid of ultra high resolution in mass detection and novel data processing method, this example demonstrates that this platform is practical for ultra high-throughput shotgun lipidomics. METHODS

Preparation and derivatization of lipid extracts from human plasma samples

[0085] All purified lipids used for internal standards were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL), Matreya, Inc. (Pleasant Gap, PA), or Nu Chek, Inc (Elysian, MN). Human plasma samples were obtained from the blood bank of NIST who collected plasma from 100 healthy, over-night fasting individuals with equal genders at 40 to 50 years of age and were stored at -80°C after received. Protein assay on the plasma samples was performed by using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL) with bovine serum albumin as a standard. All determined lipid levels were normalized to the protein content. Lipid extracts from 50 iL plasma each were separately prepared. The lipid extracts were reconstituted with a volume of 500 μΐ/mg of protein in 1 : 1 CHCls/MeOH. The lipid extracts were finally flushed with nitrogen, capped, and stored at -20 °C for ESLMS (typically analyzed within one week). A quarter of each lipid extract was reacted with CD₃I.

Mass spectrometric analysis of human plasma lipid extracts

[0086] Lipid extracts were diluted in 1 :2:4 (chloroform:methanol:isopropanol, v/v/v) to a concentration of approximately 50 ριηοΙ/μΕ and loaded into an 96-well uniplate (250 μί, Whatman, NJ). The plate was sealed with a piece of aluminum foil after sample loading to prevent the solvent evaporation. MS analysis was performed on a quadrupole-orbitrap mass spectrometer (Thermo Scientific Q-Exactive, San Jose, CA) equipped with an automated nanospray device (TriVersa NanoMate, Advion Bioscience Ltd., Ithaca, NY). An ionization voltage of 1.2 kV and a gas pressure of 2.0 psi on the NanoMate apparatus were employed for MS analysis.

[0087] The device was controlled by Chipsoft 8.3.1 software. The S-Lens RF level of the Q- Exactive source was optimized with a mixture of lipid standards, and a 75.0 (arbitrary unit) was used. Capillary temperature of the source was set to 200 °C. All other available source parameters were set to zero. The automatic gain control targeted for both full MS and AIF scans was set to lxlO⁶ ions with maximal injection time of 120 ms. The Q-Exactive was calibrated daily as recommended by the manufacturer.

[0088] To improve the mass accuracy, lock masses were used during data acquisition. A list of calculated accurate masses of lipid internal standards and commonly-occurred major lipid species were used as the locked mass list in the global parameter of acquisition method file. The Q- Exactive was set to a maximal resolution of 140,000 with various microscans (1 to 6). The full MS scan range was set according to the targets of lipid species and ESI polarity as specified. The scan range for AIF mass spectra was set from m/z 50 to the maximal value used for the corresponding full MS scan with the quadrupole set to pass all ions corresponding to the mass range of the full MS scan. Data was acquired in both positive- and negative-ion modes to match the distributed samples in the plate. Each acquisition method was programmed to acquire a full MS spectrum and three AIF spectra with three normalized collision energies of 15, 25, and 35.

Results

[0089] Lipid analysis is unique. All lipid samples analyzed can be prepared through organic solvent extraction. This procedure eliminates all non-lipid candidates of isomeric species.

Selective ionization which is comparable to electrophoretic separation can further eliminate the detection of lipid species containing any unfavorable charge propensity. Then an accurate mass of an ion, if it matches with the true mass value of a species of a lipid class possessing the charge propensity to an accuracy of approximately 0.5 ppm, would essentially identify the ion as a species of the class to a very high degree.

[0090] The structure(s) of this ion, which are still not definitively identified at this moment, can be identified by searching for the existence of its (or their) unique NL fragmentation pattern(s) present in the corresponding AIF mass spectrum since it is recognized that NL scanning is molecular species specific (FIG. 1A). Specifically, if this accurate mass-matched ion represents a species of the class, the specific NL fragmentation pattern yielding from this species must exist in the AIF mass spectrum; if such a pattern does not exist, this species is not true even accurate mass matches well with a true value. If the accurate mass of this ion matches with those of multiple isomeric species of a class, all the NL fragmentation patterns of those isomeric species should exist in the AIF mass spectrum. The regiospecificity of the paired fatty acyl chains in an identified diacylglycerol (DAG)-derived species is determined utilizing the ratio of ion intensities of the fragments corresponding to the NL of the fatty acids (FAs).

[0091] Next, what should be determined is whether the full intensity of an ion represents the abundance of the identified species described above, but not from other hidden isobaric component(s) from other lipid classes under the condition of ultra high mass resolution. The developing method calculates an intensity ratio of M+l isotopologue and monoisotopic peak in the full mass spectrum. A ratio that equals to the theoretical value of the identified species indicates that an isobaric species does not co-exist. A ratio that is less than the theoretical value indicates the existence of isobaric species and the method uses the intensity of the M+l isotopologue to derive the monoisotopic ion intensity for quantification. A ratio that is greater than the theoretical value, which is rare, but could be present, indicates the existence of an isobaric species with the M+l isotopologue. In this case, the method employs the intensity of monoisotopic peak for quantification.

[0092] Finally, the mass level of individual identified ion corresponding to a lipid class of interest is determined by ratiometric comparison of the ion intensity to that of the selected internal standard of the class detected in the full mass spectrum. A wide linear dynamic range for quantification can be present by ratiometric comparison utilizing ion intensities detected in the full mass spectrum under certain conditions. These conditions include (1) at a low lipid concentration (to avoid lipid aggregation); (2) for a polar lipid class (to minimize the effects of differential acyl chain contribution); (3) after ¹³C de-isotoping for monoisotopic peak comparison as well as to resolve any overlap between an m+2 isotopomer of an ion A with an ion containing one double bond less than the ion A; and (4) only utilizing a full mass spectrum to avoid any differential fragmentation of different molecular species of the class. The composition of the isomeric species of an ion (if present) is estimated from the intensity ratio of fragments corresponding to the NL of either sn-1 or sn-2 FAs.

The Analytical Platform

[0093] In the current platform, a lipid extract of a biological sample was first derivatized with deuterated methyl iodide (CD₃I) to convert ethanolamine glycerophospholipid (PE), lyso PE (LPE), N- monomethyl PE (MMPE), and Ν,Ν-dimethyl PE (DMPE) to d₉-choline

glycerophospholipid (dg- PC), dg-LPC, d6-PC, and d3-PC, respectively (FIG. 2). Employing this procedure is to exploit the unique NL fragmentation patterns of lithiated PC and LPC species for the analysis of ethanolamine-containing lipid species with the ANLA-SL platform since these unique NL fragmentation patterns allow definitive identification of individual molecular species of choline-containing lipid classes (see below).

[0094] It is noted that the yield of the chemical reaction does not significantly affect the analysis of these lipid classes due to the presence of an internal standard for each individual lipid class. However, this example optimized the experimental conditions to achieve a nearly complete reaction. It should also be noted that in addition to the derivatization with CD₃I, other chemical derivatization/reaction could also be used to prepare a sample for the analysis of a particular category of lipid classes (e.g., alkaline methanolysis for the enhanced analysis of sphingolipids if necessary).

[0095] Each prepared sample was appropriately distributed to two separate wells of a Teflon- coated plastic plate for sample injection with a Triversa Nanomate device. A small amount of LiOH (~50 pmol^L) was added to one of the wells. The lipid solution in this well was used for MS analysis in the positive-ion mode and the other well was for the negative-ion mode. This procedure was designed to achieve a selective ionization of different lipid classes (FIG. 2).

[0096] In each ionization mode, one full mass spectrum (ranged from m/z 350 to 1000 which covers majority of the lipid classes of interest) and an AIF mass spectrum ranged from m/z 50 to 1000 were acquired (FIG. 2). For the AIF MS analysis, the quadrupole was set to pass all ions ranged from m/z 350 to 1000 (called as the Q-window). Five full mass and 10 AIF scans at a speed of 4 Hz were acquired and averaged for each full mass or AIF mass spectrum. Accordingly, all the mass spectra necessary for lipidomics analysis can theoretically be done in 4 seconds. It should be pointed out that to eliminate ion suppression to a certain degree, the mass range could be segmented from m/z 350 to 500 and m/z 500 to 1000 for full mass spectra and from m/z 50 to 500 (with the Q-window set between m/z 350 to 500) and m/z 50 to 1000 (with the Q-window set between m/z 500 to 1000) for AIF mass spectra. The former mass range can be used for the analysis of lipid intermediates (e.g., lysolipids and acylcarnine). The spectra can be simplified and enhanced with reduced ion suppression and decreased interference from the ions present in the higher mass range which covers abundant individual molecular species of majority of phospholipid classes.

[0097] Finally, the acquired mass spectra were processed utilizing an ANLA-SL program developed in-house to identify and quantify individual molecular species of lipid classes listed (FIG. 2). The details of the method are provided below.

ANLA-SL

[0098] The workflow of the method is illustrated in FIG. 3. The method creates an in situ database of the true mass values of individual species of a lipid class of interest based on two variables: numbers of carbon atoms (m) and numbers of double bonds (n). The method also extracted the masses and intensities of each individual ions determined from the full mass spectra. Filtering of the detected masses of ions with theoretical true values of the species of a class of interest in a mass accuracy of 1 ppm yielded a list of the detected molecular species candidates of the class.

[0099] At this stage, the method searched the data set of the corresponding AIF mass spectrum to the class (based on selective ionization and Q-window) for the specific NL fragmentation pattern of each candidate. Since after solvent extraction, selective ionization, and accurate mass matching, these candidates essentially represent the species of the class. Existence of a specific NL fragmentation pattern corresponding to each candidate (whether it is isomeric to others or not) definitively identifies the species, representing that the ion in the full mass spectrum yielding the fragmentation pattern at least contain the species as a component. Accordingly, by using this approach, all individual molecular species of the class can be identified and then quantified by the developing ANLA-SL platform (see below for individual lipid class). Previous studies have demonstrated that fragment ion intensities significantly depend on the chemical structures of individual lipid species of a class as well as the collision conditions. To maximize the presence of fragment ions, the AIF mass spectra acquired at three different collision energies were averaged to result in a final AIF mass spectrum.

[0100] A few points are important here. First, confirming the presence of individual species only can be done with a specific NL fragmentation pattern since only a NL fragmentation pattern is directly and specifically linked with a molecular ion with an accurate mass. Second, this specific NL fragmentation pattern contains at least two fragment ions to eliminate any potential artifact, one of which should be a NL fragment resulted from the aliphatic chain if the species contain two aliphatic chains. Third, it is important to have an accurate mass of the NL to ensure that the fragmentation pattern is specific to the species; an ultra high resolution/mass accuracy instrument is critic yield accurate NL masses. Four, the paired NL FAs of a species yielding a specific NL fragmentation pattern was predicted utilizing the method based on the total numbers of carbon atoms and double bonds of the FAs. Finally, the intensity ratios of the monoisotopic peak vs. M+l isotopologue can provide useful information about the identification and quantification of the species. The ratios of the fragments corresponding to the neutral losses of fatty acyl chains can be used to estimate the isomeric composition of an ion and to determine the regiospecificity of individual species.

[0101] Once the candidates were identified, quantification of these species was straightforward by ratiometric comparison of the peak intensity of the ion in full mass spectrum with that of the selected internal standard of the class after C de-isotoping which was performed based on the intensity of either monoisotopic peak or M+l isotopologue. If the ion contains isomeric species, the composition of these isomeric species was determined from the intensity ratio of fragments corresponding to the neutral losses of sn-1 FAs of the isomeric species. A similar ratio should also be obtained from the fragments corresponding to the loss of sn-2 FAs of these isomeric species. The method calculated the composition by averaging both ratios.

Analysis of PC species present in human plasma lipid extracts

[0102] The PC class can be classified into three subclasses, i.e., diacyl PC (dPC), plasmenyl PC (pPC) containing a vinyl ether-linked aliphatic chain (or called alkenyl chain), and plasmanyl PC (aPC) containing an ether-linked aliphatic chain (or called alkyl chain), both at the sn-1 position of glycerol. These subclasses yield subclass-specific fragmentation patterns and follow their unique distribution rules. Any dPC species after CID yields a specific NL fragmentation pattern containing NL59.07350 (corresponding to trimethylamine), NL183.06604 (phosphocholine), NL189.07422 (lithium cholinephosphate), NL(59.07350 plus sn-1 FA mass), and NL(59.07350 plus sn-2 FA mass) (FIG. 4A). In addition, dPC species can readily be distinguished from pPC and aPC species with the accurate masses. Any pPC species can be resolved from isomeric aPC species by the fragmentation pattern with the presence of a different fragment corresponding to NL (59.07350 plus sn-2 FA mass). Accordingly, the developing platform allows identification of all PC species as described in the last subsection.

[0103] For instance, this example analyzed PC species present in lipid extracts of human plasma samples by a Q-Exactive mass spectrometer followed by utilizing an in-house ANLA-SL method. A full mass spectrum of a diluted lipid extract in the presence of a small amount of LiOH ranged from m/z 500 to 1000 was acquired in the positive-ion mode. Searching accurate masses of theoretical PC species marched with 60 ions with a total of 45 candidate PC species containing isomers. The potential isomers with different FAs were derived from the total numbers of carbon atoms and double bonds present in FAs based on the accurate masses. Each of these candidate PC species was confirmed by searching the presence of a specific NL fragmentation pattern to the individual species as outlined above (FIG. 4B). This procedure eliminated 43 candidate species to confirm the presence of a final number of 72 PC species. The

regiospecificity of each species was determined based on the intensity ratio of fragments corresponding to the losses of sn-1 and sn-2 FAs. [0104] The mass levels of individual ions were determined by comparison of the abundance of individual ions (i.e., ratiometric comparison) with that of the selected PC internal standard (i.e., di 14 : 1 PC). The composition of isomeric species of an ion was determined based on the abundance of the fragments resulted from the NL of FAs as aforementioned. Accordingly, a total of 72 PC species (including their isomers, regiospecificity, FA identities, etc.) from three subclasses present in human plasma lipid extracts were identified and quantified by the ANLA- SL platform. It should be pointed out that the isomers due to the presence of different locations of double bonds are not considered in the study.

Analysis of individual molecular species of other lipid classes present in human plasma lipid extracts in the positive-ion mode

[0105] Similar to the PC species as described above, lithiated LPC species also display a specific NL fragmentation pattern of individual species. Specifically, any lithiated LPC species after CID yields a specific NL fragmentation pattern containing NL59.07350 (i.e.,

trimethylamine), NL183.06604 (phosphocholine), and NLl 89.07422 Da (lithium

cholinephosphate). The aliphatic chain of individual LPC species can be directly derived from the accurate mass of a molecular ion. The location of the fatty acyl chain (i.e., regiospecificity) of acyl LPC can be assessed based on the intensity ratios of the fragments corresponding to

NL59.07350 and NLl 89.07422. A ratio of 2.51 or 0.33 for sn-1 or sn-2 acyl LPC, respectively, is present. Thus, similar to the identification and quantification of PC species, a total of 13 individual LPC species in human plasma lipid extracts were identified and quantified by the ANLA-SL platform.

[0106] The molecular species of PE, MMPE, DMPE, and LPE were converted to dg-, de-, d^- PC, and dg-LPC, respectively, after reaction with CD₃I as previously described. Therefore, the molecular species of these classes were identified and quantified essentially identical to PC and LPC, respectively, as described above except that the neutral losses of 59.07350, 183.06604, 189.07422, 59.07350 plus sn-1 FA mass, and 59.07350 plus sn-2 FA mass shifted the neutral losses with additional 9, 6, or 3 deuterium masses..

13

[0107] Sphingomyelin (SM) species overlap with m+1 C isotopologues of PC species in low- resolution mass spectrometry whereas these species are well resolved in high-resolution mass spectrometry. For example, the molecular weight of dl 8: 1/C18:0 SM is 737.61433Da while that

13

of m+1 C isotopologues of dil6: l PC is 737.55258 Da; their mass difference is 83.7 ppm. Previous extensive characterization of purified sphingomyelin (SM) species has demonstrated a specific NL fragmentation pattern resulted from lithiated SM species. These characteristic fragments include NL59.07350 (i.e., trimethylamine), NL183.06604 (phosphochohne),

NL213.07661 (phosphochohne plus methoxyl), and a NL fragment resulted from sphingoid base

(e.g., NL429.30080 for sphingosine and NL431.31645 for sphinganine). Particularly,

NL213.07661 is sensitive and unique to the lithiated SM species. Therefore, SM species can be readily identified and quantified by the ANLA-SL platform.

[0108] Similar to SM species, the presence of a specific NL fragmentation pattern resulted from lithiated HexCer species has been previously demonstrated from extensive characterization of purified hexosylceramide (HexCer) species (including both galactosylceramide and

glucosylceramide). These characteristic fragments include NL162.05282 (i.e., monohexose derivative), NL180.06339 (162.05282 plus H₂0), NL210.07395 (180.06339 plus methyl aldehyde), and a NL fragment resulted from sphingoid base (e.g., NL418.29305for sphingosine and NL420.30870for sphinganine). In the case of HexCer, NL210.07395 is sensitive and unique to the lithiated HexCer species.

[0109] The ANLA-SL platform can also be used to identify and quantify some low abundance lipid classes in the positive-ion mode as their lithium adducts. For example, tandem MS analysis of lithiated acylcarnitine species after CID displayed a specific NL fragmentation pattern (FIG. 5A). The abundant fragment ions include NL59.07350 (i.e., trimethylamine), NL143.09463 (i.e., carnitine), and NL (59.07350 plus FA mass). The ANLA-SL platform utilizing this fragmentation pattern in combination with accurate masses of lithiated acylcarnitines detected by the Q- Exactive mass spectrometer allowed identification and quantification of 11 acylcarnitine species from human plasma lipid extracts. Similarly, a specific NL fragmentation pattern of lithiated lysoSM species after CID was also determined (FIG. 5B). The resultant fragment ions include NL17.99976 (i.e., water molecule), NL59.07350 (i.e., trimethylamine), NL183.06604

(phosphochohne), and NL212.06878 (phosphochohne plus methyl aldehyde). This example identified and quantified dl8:0 and dl8: 1 lysoSM species from human plasma lipid extracts by the ANLA-SL platform.

[0110] A few points are worth of notice. First, in addition to the lipid classes described above, the ANLA-SL platform can be further expanded to include other lipid classes if a specific NL fragmentation pattern can be defined for a particular lipid class (e.g., monohexosyl DAG and dihexosyl DAG for which specific NL fragmentation patterns from their lithium adducts have been elucidated). Second, this example employed lithium adducts through addition of LiOH since more unique NL fragmentation patterns can be resulted from lithium adducts of many lipid classes. Similar approach(es) can be employed for the platform if other cation adduct(s) can yield useful specific NL fragmentation patterns from those adducts of lipid classes. Third, many cation adducts of lipids (e.g., proton, sodium, potassium, lithium-6 isotopomer, etc.) other than lithium- 7 could be detected by a sensitive mass spectrometer. However, the presence of these adducts does not affect the identification and quantification of lipid species only based on lithium-7 adducts for polar lipid classes due to the presence of internal standard(s) of each lipid class although correction factors for individual molecular species of non-polar lipid classes have to be considered. Finally, potential overlaps of particular cation adducts with lithium adducts of lipid species are concerned in low-resolution mass spectrometry, but can be well resolved by ultra high-resolution mass spectrometry. For example, sodium adducts of pPC species or PC species with a FA chain containing odd-numbered carbon atoms overlap with some lithium adducts of diacyl PC species in low-resolution mass spectrometry since a mass difference of 16 Da between sodium and lithium is equivalent to the oxygen mass or the mass of a methylene plus two protons. All these isobaric species can be readily resolved by high- resolution mass spectrometry for the masses of 15.97377 (Na-Li), 15.99492 (O), and 16.03130 (CH₂+2H).

Analysis of individual molecular species of anionic lipid classes present in human plasma lipid extracts in the negative-ion mode

[0111] After a lipid solution was derivatized with CD₃I, any ion suppression due to the presence of abundant PE species was minimized for the analysis of anionic lipids in the negative- ion mode. Therefore, this procedure substantially simplifies the anionic lipid profile present in a mass spectrum. The ANLA-SL platform can be similarly applied to the anionic lipid classes as those in the positive-ion mode if there exists a specific NL fragmentation pattern of individual lipid species of a class.

[0112] Product-ion MS analysis after CID displayed an informative NL fragmentation pattern of deprotonated PI species. The NL fragmentation pattern was similar to that as elucidated with an ion-trap mass spectrometer. Specifically, any PI species shows a NL fragmentation pattern containing NL(sn-l FA mass), NL(sn-2 FA mass), NL(sn-l FA mass plus 162.05282), and NL(sn-2 FA mass plus 162.05282). The intensity ratios of the fragments resulted from NL of sn-2 FA vs. those from sn-1 FA loss are approximately 0.25 and 0.2 for FA loss and the loss of FA plus 162.05282, respectively. Accordingly, the specific NL fragmentation pattern as well as the fragment ion ratios allows identification and quantification of individual PI species of biological samples including isomer composition and regiospecificity by the ANLA-SL platform. A total of 22 individual PI species in human plasma lipid extracts were identified and quantified.

[0113] Although specific NL fragmentation patterns for cardiolipin (CL) and monolysoCL species are not present, their doubly-charged property and the unique existence of [M-2H+1] ^" isotopologues are very specific to these classes of species. Accurate mass search for the [M- 2H+1] ^" ions can "isolate" all the ions of CL and monolysoCL species and quantify these ions by ratiometric comparison to a selected internal standard of these species.

[0114] Product-ion MS analysis of individual ion corresponding to CL or monolysoCL species can always be employed to elucidate the acyl chains of individual species and to determine isomer composition of the ions.

[0115] Collectively, the current study developed a new, sensitive, and ultra high-throughput shotgun lipidomics technology platform for global analysis of cellular lipi domes by using ultra high-resolution/mass accuracy mass spectrometers (e.g., Q-Exactive). This platform can be expanded to analyze individual lipid species of any class as long as a specific NL fragmentation pattern of individual molecular species of the class is elucidated.

Example 2

[0116] This example demonstrates an approach for accurate mass searching by exploring one of the major features of lipidomics data that molecular species of a lipid class are present in ion clusters where neighboring masses from different species differ by one or a few double bonds. This example first opened the mass-searching window to ±13 ppm to search an entire group of species of a lipid class; these species contain an identical number of carbon atoms. Then accurate

13

mass searching of the plus one C isotopologue was used to eliminate the potential false positive

13 13

since the overlaps of the plus one C isotopologue with other C isotopologues are minimal and

13

these overlaps if present are very different from the overlaps between M+2 C isotopologues and L ions. This new mass searching approach was validated through comparing with the species determined by unit-resolution MS (i.e., multi-dimensional MS-based shotgun lipidomics (MDMS-SL)). Materials and Methods

[0117] Materials. All phospholipid species used either as internal standards or for mixing experiments were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). All the solvents were obtained from Burdick and Jackson (Honeywell International Inc., Muskegon, MI). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

[0118] Preparation of phosphatidylcholine Mixtures and Lipid Extracts from Biological Samples. The stock solutions of 16:0-18:2, 16:0-18: 1, and 16:0-18:0 phosphatidylcholine (PC) species were separately prepared in CHCVMeOH (1 : 1, v/v). The concentration of each stock solution was quantified individually by comparison with the internal standard dil4: 1 PC using ESLMS. The mixtures of the PC species were prepared from individual PC species stock solutions. The serial ratios in the mixtures were obtained by fixing the concentration of one PC species while varying the other(s). For convenience to display the overlapped ion peaks and

13 process the mass shifts, these ratios were given as those between 16:0-18:0 PC and M+2 C isotopologues of 16:0-18: 1 PC.

[0119] C57BL/6 wild type male mice (4 to 6 months of age) were originally purchased from The Jackson Laboratory (Bar Harbor, ME) and/or housed in a full barrier facility with a 12-h light/dark cycle and maintained on standard chow (Diet 5053; Purina Inc., St. Louis, MO). All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Studies Committee at Sanford-Burnham Medical Research Center at Lake Nona, Orlando, FL. Mice were sacrificed by asphyxiation with CO₂. Mouse plasma and tissue samples were harvested immediately after sacrifice. Plasma sample (100 μί) from each animal was used to prepare the plasma lipid extracts by using a modified Bligh and Dyer procedure. The final concentration of each lipid extract was reconstituted in 1 mL of 1 : 1 (v/v) CHCl₃/MeOH. Lipid extracts of mouse tissue samples were prepared. The lipid extracts were finally flushed with nitrogen, capped, and stored at -20 °C for ESLMS analyses.

[0120] MS Analysis of PC mixtures and lipid extracts of biological samples. High mass accuracy/resolution MS analyses of lipids were performed by using a Q-Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an automated nanospray apparatus (Advion Bioscience, Ithaca, NY). The NanoMate apparatus was controlled by Chipsoft 8.3.1 software. An ionization voltage of 1.4 kV and a gas pressure of 0.25 psi were employed for the MS analyses. All mass spectra were recorded under Xcalibur 2.2.48 software with an AGC value of 1 x 10⁵ in the full MS mode for up to 10 s, in which the mass resolution of the analyzer was set at 140,000, 70,000, or 30,000 (ra/Am, fwhm at m/z 400) as indicated.

[0121] MDMS-SL was performed by using a TSQ Quantum Ultra mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Individual PC mixtures or lipid extracts were diluted to approximately 50 pmol^L in CHCl₃/MeOH/isopropanol (1 :2:4, v/v/v) or less prior to infusion with the Nanomate device.

Results

[0122] Recognition of the mass shift resulted from the double bond overlapping effect analyzed by high mass accuracy/resolution shotgun lipidomics. When this example calculated the mass

13

defects of natural elements relative to C atom (FIG. 6A-B), it was recognized that the partial

13

overlap of the two C atom-containing isotopologue (i.e., M+2 isotopologue) of a species M with the ion of a species L containing a less double bond (i.e., the double bond overlapping effect). Although isotopologues resulting from chlorine-37 and sulfur-34 could also overlap with

13

the M+2 C isotopologue, chlorine (which can be easily probed due to the natural abundance of

37 Cl at 31.9784%) and sulfur-containing lipids are less common than C, H, O, N, and P- containing lipids, particularly in the mammalian system. Therefore, the double bond overlapping effect would be the major and most dominant effect contributing to the mass shift and complicating the isotopic patterns of lipid species in high mass resolution shotgun lipidomics. More complication of this overlapping effect was its dependency on the mass resolution of the employed instrument and the molar ratio of the overlapped ions.

[0123] Mass shift resulted from the double bond overlapping effect is dependent on the mass resolution of an instrument and the molar ratio of the overlapped ions. To demonstrate the double bond overlapping effect-induced mass shift, this example simulated the expanded mass spectra

13

displaying the ion peaks of the M+2 C isotopologue and the ion of the species L containing one less double bond than M at an equal intensity ratio for the species of PC and phosphatidylinositol (PI) classes (FIG. 7). This example found the resolution of these ion peaks depended on the mass resolution of the instrument. Specifically, the two ions essentially collapsed into a single, broad peak at an instrumental mass resolution of 75K (which corresponds to the mass resolution of the Q-Exactive mass spectrometer at this mass region) or less (FIG. 7A and D). These ions were partially resolved at the instrumental mass resolution of 150K at the mass region (FIG. 7B and E) and could only be totally resolved with an instrumental mass resolution of 600K which is unachievable at the current time (FIG. 7C and F).

[0124] Due to this overlap, the apex of this collapsed peak was shifted to the middle of these ions if the instrumental mass resolution is at 75K or less. The location of the apices of the collapsed peaks could shift from the ion of L when the species M is absent to the place of 12.78 ppm downside relative to the ion of L when the species L is absent. This location entirely depends on the intensity (i.e., molar) ratio of the species L and the M+2 isotopologue. Therefore, the requirement of instrumental mass resolution to resolve these ions also varies with the molar ratios.

[0125] To demonstrate the dependence of the overlapping ion peak on the molar ratio of the L and M+2 ions, this example prepared a series of mixtures containing two standard PC species of which the difference between the two fatty acyl chains was one double bond (i.e., 16:0-18: 1 PC (M) and 16:0-18:0 PC (L)). This example determined the mass shift of the apex of the overlapping peak of M+2 13 C isotopologue and L ion as varied with the molar ratios of M+2 and L ions (FIG. 8). Essentially identical to the simulation, the mass shift of the overlapping ion peak depended on the molar ratios of the mixtures under experimental conditions (FIG. 8).

[0126] Direct accurate mass searching by using instrumental mass accuracy leads to the substantial miss of species. The mass shift due to the double bond overlapping effect alone apparently also complicates the direct accurate mass searching for identification of lipid species, thereby leading to a significant false negative result. For example, the Q-Exactive mass spectrometer possesses a mass accuracy of approximately 1 ppm according to the manufacturer's specification. However, only 10% of lipid species after direct infusion in comparison to those identified by MDMS-SL were hit when accurate mass searching with a mass window of ± 1 ppm was conducted. These hit species largely corresponded to those which were abundant and not

13

overlapped with other lipid species from other lipid class or M+2 C isotopologues of the identical class.

[0127] In addition to the substantial miss of species due to the double bond overlapping effect, this example found a mass searching window of ± 3.5 ppm was necessary despite the utility of a lock mass to increase the coverage of species in comparison to those lipid species identified by MDMS-SL considering the potential ionization instability. Based on these findings, a mass- searching window from -13 ppm to 3.5 ppm of each theoretical mass should be used to search all the species. Unfortunately, accurate mass searching with such a wide mass window could result in a significant number of false positive hits.

[0128] The method searched the candidate species as a group in which all the species contained an identical number of carbon atoms, but with various numbers of double bonds. This example first searched a species M of the group containing a possibly highest number of double bond in the group with a mass searching window ± 3.5 ppm of the theoretical mass of the interesting species. If a hit was found, this example then checked the presence of its M+l isotopologue also with the mass searching window of ± 3.5 ppm. If this isotopologue did not exist, this potential candidate species was discarded. If this species was validated with the presence of its M+l isotopologue, this example then determined its double bond overlapping effect to the species containing one less double bond. The double bond overlapping effect was determined by using accurate mass searching window from -13 ppm to 3.5 ppm to search for the presence of L species which was further validated with the presence of L+l isotopologue within the accurate mass searching window of ± 3.5 ppm.

[0129] This procedure was repeated till the species containing no double bond to determine all the candidate species of this group. This example found that the majority of the false positive ions were eliminated after searching the presence of the M+l isotopologues while the false negative results were avoided after considering the double bond overlapping effect.

[0130] This example performed analysis of numerous lipid extracts from biological samples by using this approach and compared the obtained results to those identified by MDMS-SL. This example found that all the species identified by MDMS-SL were present in the dataset obtained from the accurate mass searching, indicating the elimination of false negative results. This example also found that there existed many extra species in very low abundance in comparison to those identified by MDMS-SL. This difference was likely resulted from the better sensitivity and dynamic range possessed by the high mass accuracy/resolution mass spectrometer than that possessed by the unit resolution instrument. Occasionally, this example also detected a few extra species (< 1%) which were present in modest abundance obtained from the high accurate mass searching in comparison to the dataset obtained from the MDMS-SL analysis. It was believed that these extra species were due to the false positive searching and have to be eliminated by tandem mass spectrometry which should ultimately be performed to identify the identities of the fatty acyl chains and the regioisomers of those obtained species from high accurate mass searching. [0131] Finally, one could extend two interesting points from the current study. First, it would be more accurate to extract peak areas than directly measure the peak heights due to the presence of peak broadening resulted from the double bond overlapping effect in high mass accurate shotgun lipidomics. Second, accurate quantification needs vigorous determination of linear dynamic range, repeatability, limit of quantification, etc., the dataset of the extracted peak areas has indeed set up a foundation for quantification of those candidate species determined through high accurate mass searching, particularly in combination with the deconvolution of 13 C isotopologue patterns which were already conducted in the high accurate mass searching approach.

[0132] The double bond overlapping effect dominates the full scan mass spectra in high mass accurate shotgun lipidomics. This example provides the evidence from both simulation and experimental data that (1) direct accurate mass searching of high resolution mass spectra could result in a substantial number of false negative hits if a specified instrumental mass accuracy was used for searching and (2) substantial numbers of false positive species would be included if the mass searching window was opened even to ± 3.5 ppm. This complication has not been recognized and this study is the first to address this issue. It is also noted that the double bond overlapping effect could also complicate the high mass accurate MS-based LC-MS efforts of lipidomics/metabolomics due to the difficult resolution of species differing only by one or two double bonds without extra efforts.

A A A

[0133] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0134] The methods illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the invention embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0135] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the methods. This includes the generic description of the methods with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0136] Other embodiments are within the following claims. In addition, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

CLAIMS:

1. A method for identifying a plurality of lipids in a sample, comprising:

subjecting a first portion of the sample to mass spectrometry to obtain a full mass

spectrum comprising a plurality of detected full mass values;

subjecting a second portion of the sample to mass spectrometry to obtain an all-ion- fragmentation (AIF) mass spectrum comprising a plurality of detected ion fragment mass values;

identifying, for each detected full mass value, one or more candidate lipids having a

matched theoretical mass value, from a database comprising a plurality of lipids, a list of ion fragments for each of the lipids, and a theoretical mass value for each of the lipids and each of the ion fragments;

retrieving from the database, for each of the candidate lipids, the corresponding ion

fragments and theoretical mass values of the fragments; and

determining that if the plurality of detected ion fragment mass values include values that match the theoretical mass values of the corresponding ion fragments of one or more of the matched lipids, then the one or more of the matched lipids are present in the sample,

thereby identifying a plurality of lipids in the sample.

2. The method of claim 1, wherein the corresponding ion fragments of each candidate lipid comprise at least two neutral loss (NL) fragments of the candidate lipid.

3. The method of claim 1 or 2, wherein the AIF mass spectrum comprises results from at least two scans at different collision energies.

4. The method of any preceding claim, wherein the AIF mass spectrum comprises a positive-ion mode mass spectrum.

5. The method of any preceding claim, wherein the AIF mass spectrum comprises a negative-ion mode mass spectrum.

6. The method of any preceding claim, wherein the full mass spectrum covers at least a range of 500-800 mass charge (m/z) ratios.

7. The method of claim 6, wherein the full mass spectrum covers at least a range of 350- 1000 m/z ratios.

8. The method of any preceding claim, wherein the AIF mass spectrum covers at least a range of 200-800 m/z ratios.

9. The method of claim 8, wherein the AIF mass spectrum covers at least a range of 50- 1000 m/z ratios.

10. The method of any preceding claim, further comprising derivatizing the sample prior to mass spectrometry.

11. The method of any preceding claim, further comprising enriching the content of lipids in the sample with organic solvent extraction or chromatography.

12. The method of any preceding claim, wherein the sample is a biological sample.

13. The method of claim 12, wherein the biological sample comprises a body fluid.

14. A method for identifying a plurality of lipids in a sample, comprising:

receiving, at a computer, a full mass spectrum comprising a plurality of detected full mass values, generated from the sample;

receiving an all-ion- fragmentation (AIF) mass spectrum comprising a plurality of

detected ion fragment mass values, generated from the sample; identifying, for each detected full mass value, one or more candidate lipids having a matched theoretical mass value, from a database comprising a plurality of lipids, a list of ion fragments for each of the lipids, and a theoretical mass value for each of the lipids and each of the ion fragments;

retrieving from the database, for each of the candidate lipids, the corresponding ion fragments and theoretical mass values of the fragments; and

determining that if the plurality of detected ion fragment mass values include values that match the theoretical mass values of the corresponding ion fragments of one or more of the matched lipids, then the one or more of the matched lipids are present in the sample,

thereby identifying a plurality of lipids in the sample.

15. A method for identifying the composition of a mass spectrum peak, comprising:

subjecting a sample comprising a plurality of lipids to mass spectrometry to obtain a mass spectrum comprising at least a mass spectrum peak;

identifying a first lipid that is suspected to be present in the peak;

generating a list of lipids that have identical number of carbons as the first lipid but have more or fewer double bonds than, or have the same number of double bonds as, the first lipid;

determining whether the peak is an aggregate of two or more peaks formed by lipids from the list.

16. The method of claim 15, further comprising comparing the peak to the ¹³C isotopologues

13

of the lipids in the list to determine whether any of the C isotopologues is present in the peak.

17. A method for identifying the composition of a peak in mass spectrum, comprising:

receiving, at a computer, a mass spectrum generated from a sample comprising a plurality of lipids, wherein the mass spectrum comprises at least a mass spectrum peak; identifying a first lipid that is suspected to be present in the peak;

generating a list of lipids that have identical number of carbons as the first lipid but have more or fewer double bonds than, or have the same number of double bonds as, the first lipid;

determining whether the peak is an aggregate of two or more peaks formed by lipids from the list.

13

18. The method of claim 16, further comprising comparing the peak to the C isotopologues

13

of the lipids in the list to determine whether any of the C isotopologues is present in the peak.