Systematic Screening for Novel Lipids by Shotgun Lipidomics

Identification and Biological Activity of Synthetic Macrophage Inducible C-Type Lectin Ligands. Chriselle D. Braganza , Thomas Teunissen , Mattie S. M...
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Systematic Screening for Novel Lipids by Shotgun Lipidomics Cyrus Papan,† Sider Penkov,† Ronny Herzog,†,‡ Christoph Thiele,§ Teymuras Kurzchalia,† and Andrej Shevchenko*,† †

Max Planck Institute for Cell Biology and Genetics, Pfotenhauerstraße 108, 01307 Dresden, Germany Lipotype GmbH, Dresden, Saxony 01307, Germany § LIMES Life and Medical Sciences Institute, University of Bonn, Carl-Troll-Strasse 31, 53115 Bonn, Germany ‡

S Supporting Information *

ABSTRACT: A commonly accepted LIPID MAPS classification recognizes eight major lipid categories and over 550 classes, while new lipid classes are still being discovered by targeted biochemical approaches. Despite their compositional diversity, complex lipids such as glycerolipids, glycerophospholipids, saccharolipids, etc. are constructed from unique structural moieties, e.g., glycerol, fatty acids, choline, phosphate, and trehalose, that are linked by amide, ether, ester, or glycosidic bonds. This modular organization is also reflected in their MS/MS fragmentation pathways, such that common building blocks in different lipid classes tend to generate common fragments. We take advantage of this stereotyped fragmentation to systematically screen for new lipids sharing distant structural similarity to known lipid classes and have developed a discovery approach based on the computational querying of shotgun mass spectra by LipidXplorer software. We applied this concept for screening lipid extracts of C. elegans larvae at the dauer and L3 stages that represent alternative developmental programs executed in response to environmental challenges. The search, covering more than 1.5 million putative chemical compositions, identified a novel class of lysomaradolipids specifically enriched in dauer larvae.

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tissues are directly infused into a tandem mass spectrometer at a nanoliter-range flow rate. Infusing a few microliters of analyte supports continuous spraying for more than 30 min and acquiring hundreds of MS and MS/MS spectra in both positive and negative mode.16 Informative high resolution MS/MS spectra acquired at the millisecond time scale enable the structural characterization of all ionized species either by datadependent acquisition (DDA) or with the aid of an inclusion list by targeted MS/MS (t-MS2). Since the practical width of the precursor isolation window allowed by quadrupole or linear ion trap analyzers typically exceeds ±0.5 Th (ref 17), the analysis can be set up such that within a m/z range of several hundreds of Th each lipid precursor will be fragmented. Thus, a shotgun t-MS2 experiment performed on a total lipid extract using a high resolution instrument yields a spectra data set containing accurate masses and abundances of all detectable precursors and all their fragments. One should, however, consider that dozens of isobaric precursors with strongly varying abundances may be transmitted through an isolation window of 1 Th (ref 18) during a MS/MS experiment. Therefore, shotgun tandem mass spectra are heavily convoluted

ipids are structurally diverse molecules involved in a wide range of biological functions such as cell structure and compartmentalization, energy storage, and signaling (reviewed in refs 1−5). Conveniently, the LIPID MAPS consortium has divided all known lipids into eight major categories6 and over 550 classes have been recognized. Despite their compositional diversity, complex lipids are composed of a rather limited set of structural moieties, such as fatty acids, glycerol, ethanolamine, sphingoid bases, and carbohydrates, to mention only a few. Usually, they are joined by ester, ether, amide, or glycosidic bonds, leading to a generally predictable fragmentation behavior7 under low energy collision-induced dissociation (CID). For example, CID of molecular cations of ceramides produce the characteristic long chain base fragments (reviewed in ref 8). Molecular anions of lipids comprising fatty acid moieties (e.g., glycerophospholipids) yield corresponding carboxylate fragments or products of their neutral losses as ketenes (reviewed in refs 9−11). The detection of shared structural moieties is implemented in shotgun profiling of lipid extracts by precursor and neutral loss scanning,10,12−14 although their abundance may vary in a lipid structure-dependent manner and other lipid class specific fragments may be produced concomitantly.15 Shotgun lipidomics (reviewed in refs 10 and 12) is an analytical approach in which total lipid extracts of cells or © 2014 American Chemical Society

Received: December 16, 2013 Accepted: January 28, 2014 Published: January 28, 2014 2703

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fractions of the total extract were collected by chromatography on a silica gel column (Kieselgel 60, 0.04−0.063 mm, Carl Roth GmbH, Karlsruhe, Germany) equilibrated with chloroform by two-step elution with neat chloroform and methanol. The methanol fractions, enriched with polar lipids, were pooled and concentrated for further analysis. Mass Spectrometry. Ten μL of sample extract were mixed either with 10 μL of 13 mM ammonium acetate in isopropanol or with 10 μL of 0.05% (v/v) triethylamine in methanol in a 96 well plate (Eppendorf, Hamburg, Germany). Samples were infused via robotic nanoflow electrospray ionization (ESI) source Triversa NanoMate (Advion BioSciences, Ithaca NY) into a hybrid quadrupole Orbitrap tandem mass spectrometer Q Exactive, (Thermo Fisher Scientific, Waltham, MA). The NanoMate was controlled by Chipsoft 8.3.1 software; backpressure was 0.8 psi and ionization voltage was 1.2 kV in negative mode. Ion transfer tube temperature was set to 200 °C and S-Lens level to 50. Full MS spectra were acquired under the targeted mass resolution Rm/z 200 = 140 000 (full width at half maximum at m/z 200), target value for the automated gain control (AGC) of 1 × 106, and maximum ion injection time of 50 ms. An abundant background peak of octadecyl(di-tertbutylhydroxyphenyl) propionate with m/z 529.462621 was used as a lock mass. MS/MS spectra were acquired with the target mass resolution Rm/z 200 of 70 000, target AGC value of 1 × 105, and maximum ion injection time of 1000 ms. In t-MS2 experiments, the width of precursor isolation window was set to 1 Th and was centered on each half integer m/z (e.g., 400.5; 401.5; ...) by using an inclusion list covering the m/z range of 500.5 to 1100.5. Normalized collision energy was set to 25%. Data Processing. Spectra import and basic operational features of LipidXplorer software were applied as described.21 Note that the check-box “No permutations” located at the “Run” panel should remain unchecked.

and comprise fragments originating from all co-isolated precursors and from the chemical background. Therefore, even the identification of known lipid molecules by screening against a library of reference spectra is often ambiguous and unexpected or unknown molecules will be overlooked. Here, we report on a methodology for the systematic discovery of novel lipids by shotgun lipidomics. It relies upon two intuitive rationales: first, we assumed that in shotgun experiments precursors of both known and unknown lipids are ionized and fragmented. However, the latter might remain unrecognized by conventional data mining routines that are biased toward identification of anticipated lipid classes and species. Second, species of currently unknown lipid classes might still resemble known lipids and comprise the same or similar structural moieties (“building blocks”) that could be recognized by the interpretation of MS/MS spectra with dedicated software.19−21 Here, we describe the design of experiments and a data processing routine that allow screening for species of unknown lipid classes by covering a very large number of assumed structures. Our findings demonstrate the high specificity of the shotgun screening methodology and its potential for the discovery of new lipids by structural homology-driven lipidomics.

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MATERIALS AND METHODS Chemicals, Solvents, and Lipid Standards. Common chemicals and solvents of ACS or LC−MS grade were from Sigma−Aldrich Chemie (Munich, Germany) or Fluka (Buchs St. Gallen, Switzerland); methanol (LiChrosolv grade) was from Merck (Darmstadt, Germany). Synthetic lipid standards were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Yeast Lipid Extracts Spiked with Synthetic PC. Ca. 5 × 106 cells from a frozen culture of the budding yeast strain BY4741 washed with 150 mM ammonium bicarbonate (ABC) buffer pH 8 were thawed, diluted in 500 μL of ABC buffer, and disrupted with ca. 100 μL of 0.5 mm zirconium beads (BioSpec Products, Bartlesville, OK) using a TissueLyser (Qiagen, Hilden, Germany) in a cold room. Of this lysate, 200 μL was mixed with 10 μL of internal standard containing 40 pmol of diether PC 40:0 and, where specified, with 2 μL of the mixture of clicked phosphatidylcholines (cPC) synthesized as described22 and dissolved in chloroform/methanol (2:1; v/v) at a concentration of 0.25 pmol/μL. Cell lysates were extracted by adding 1 mL of chloroform/methanol (2:1, v/v) in a shaker for 60 min at 4 °C. After centrifugation at 3000 rpm for 5 min, the lower organic phase containing the lipids was recovered, dried under vacuum in a desiccator, and redissolved in 100 μL of chloroform/methanol (2:1, v/v). Total Lipid Extract from C. elegans. To compare the lipid composition of reproductive L3 and dauer larvae, we made use of the temperature sensitive mutant daf-2(e1370) that reproduces at 15 °C but forms dauer larvae at 25 °C (ref 23). Worms were maintained at 15 °C on NGM agar plates using the E.coli strain NA22 as a food source. Embryos derived from this strain were grown at 15 °C or at 25 °C until L3 larvae or arrested as dauer larvae, respectively. At this stage, larvae were snap-frozen in liquid nitrogen and stored at −80 °C. Samples of 30000 L3 or dauer larvae in 1 mL of water were homogenized by three rounds of freezing followed by thawing in an ultrasonic bath and extracted according to the method of Bligh and Dyer24 by adding 3.75 mL of chloroform/methanol (1:2), 1.25 mL of chloroform, and 1.25 mL of water. Polar lipid

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RESULTS

Identification of Known Lipids by LipidXplorer. Central to our approach is screening of collections of shotgun spectra by LipidXplorer software.20 LipidXplorer imports all (MS and MS/MS) spectra acquired in the series of shotgun experiments into a flat-file database termed the MasterScan. Upon compiling the MasterScan, LipidXplorer associates related MS and MS/MS spectra and aligns them considering instrument-dependent peak attributes. Next, LipidXplorer employs a declarative Molecular Fragmentation Query Language (MFQL) to formalize the criteria of lipid identification: more specifically, MFQL queries describe which precursors and fragment ions should be used for recognizing the lipid species, how the identification should proceed, and how the identified species should be named and reported. Below, we briefly explain how to formulate MFQL queries for identifying species of lipid classes with exactly known structures and then extend them to screening for novel lipids. As an example, here we describe the query for identifying phosphatidylcholine (PC) species in shotgun MS and MS/MS spectra acquired in negative mode. A typical MFQL query consists of four sections: DEFINE, IDENTIFY, SUCHTHAT, and REPORT. 2704

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and one nitrogen and one phosphorus atom. The range of double bond equivalent (DBR) (ref 25) indicates that each fatty acid moiety should contain less than six double bonds in total. Precursors should be detected as singly charged anions (CHG = −1). Next, the query defines that each of the two carboxylate fragments FA1 and FA2 should contain exactly 2 oxygen atoms; however, the number of carbon and hydrogen atoms, as well as the unsaturation of hydrocarbon moieties, may vary within the specified ranges. It is not required that the MFQL query encompasses all characteristic fragments. For example, it would be possible to DEFINE and use for the PC identification also their [M − 15]− fragments (Figure 1). However, their abundance strongly depends on the applied collision energy (CE), while the abundance of carboxylate fragments of fatty acid moieties practically plateaus within the CE range of 40 to 65 eV (ref 26) making them detectable in MS/MS spectra once sufficient energy was applied. The IDENTIFY section determines in which spectra (MS or MS/MS) the respective precursors and fragments are to be searched for: the precursor mass PR in the survey (−)MS spectrum and the FA1 and FA2 fragments in the corresponding (−)MS/MS spectrum. In the SUCHTHAT section, sum formula arithmetic is applied in order to enhance the identification specificity by eliminating random combinations of fragments and precursors.20,21 The term “C10 H21 O6 N1 P1” describes a putative structural moiety that complements the sum compositions of FA1 and FA2 to the total sum composition of the intact acetate adduct of PC; as such, it is not detectable in MS/MS spectra as a fragment but is employed in the SUCHTHAT section as an additional constraint. In this way, the SUCHTHAT section distinguishes individual molecular species by identifying the combinations of fatty acid moieties matching the compositional constraints and mass of the fragmented precursor. The second SUCHTHAT condition is bound by two boolean AND operations and relates to the known PC fragmentation mechanisms. In MS/MS spectra of glycerophospholipids, the relative abundance of carboxylate fragments depends on the position (sn-1 or sn-2) of corresponding fatty acid moieties.27 It could be used to distinguish isobaric ester and ether lipids28 and to reduce the impact of chemical noise. Here, it is required that the ratio of intensities of peaks corresponding to plausible FA2 and FA1 fragments must be smaller than a factor of 5. Although in MS/MS spectra of anion adducts of PC a typical ratio of the abundances of carboxylate fragments is close to 2.5 (Figure 1; ref 26), here we applied a 2-fold larger margin to account for the possible impact of variant PC structures that were considered in further screens. Note that LipidXplorer assigns FA1 and FA2 to a pair of fragment ions matching the DEFINEd compositional constraints in an arbitrary order. If permutations of FA1 and FA2 are enabled by the spectra processing settings, LipidXplorer considers all possible combinations independently. Therefore, for consistency in our interpretation, we applied an additional clause requiring that the abundance ratio of bona f ide FA1 and FA2 fragments should be larger than 0.2 AND smaller than 5. The PC identification with the above MFQL query proceeds as follows: in the survey MS spectra, LipidXplorer identifies candidate precursors whose exact masses match the expected compositional constraints. Then, in the MS/MS spectra associated with these precursors, the software will look for the fragment ions whose masses match the compositional requirements of FA1 and FA2 and select pairs that also satisfy

In negative mode, PC are detected as molecular adducts of [M + CH3COO]− that, upon low energy CID, produce abundant carboxylate fragments from their fatty acid moieties (Figure 1).

Figure 1. (A) (−)FT MS/MS spectrum of the acetate adduct [M + CH3COO]− of PC 18:0/14:0 (inset). [R1COO]− and [R2COO]−: carboxylate ions of the fatty acid moieties R1 and R2 at the sn-2 and sn1 positions, respectively; [ChP-15]−: the fragment of choline phosphate with methyl group loss (−15 Da). (B) MS/MS spectrum of the clicked-PC (cPC). Carboxylate ions of both fatty acid moieties [R1COO]− and [R2COO]− were observed along with peaks originating from the fragmentation enhanced by the triazolylhydroxycoumarin moiety and from chemical noise.

First, the query DEFINEs the composition of PC precursors (“PR”): their elemental (also often termed as “sum”) composition constraints encompass a range of carbon and hydrogen atoms from 30 to 80 and 55 to 200, respectively, that reflect the allowed variation in the length and unsaturation of both hydrocarbon moieties; at the same time, it requires that plausible precursors should contain exactly 10 oxygen atoms 2705

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the SUCHTHAT constraints. If all these requirements are met, the lipid species is identified and will be REPORT’ed according to a user-defined format (for clarity, the REPORT section is not discussed here since it is not concerned with the identification of unknown lipids; see ref 21 for further details). To test the query, we analyzed a total lipid extract of the budding yeast. Screening a shotgun data set comprising 1550 peaks in (−)FT MS and 28 779 peaks in 400 (−)FT MS/MS spectra took 18 s on a desktop computer (Intel OctaCore i7 CPU 880; 3.07 GHz; 16 GB RAM running Microsoft Windows 7 64bit) and identified 14 PC species (Supporting Information Figure 1S), whose relative abundances corroborated the profile previously determined by an independent method of multiple precursor ion scanning.29 We emphasize that LipidXplorer only considers ions specified at the DEFINE section, while other ions (regardless whether they are originating from the same or from different molecules) are ignored. Note that lipids can usually be identified by several independent combinations of fragments. Since masses and abundances of all fragments detected in all spectra are stored in the MasterScan database, it can be successively probed with multiple independent MFQL queries with no need for reacquiring or reprocessing the spectra. Identification of Unknown Structurally Related Lipids. In MFQL queries, the elemental composition constraints are defined as ranges: in the above query, the number of carbon and hydrogen atoms is allowed to vary, while the number of heteroatoms (oxygen, nitrogen, and phosphorus) is fixed. We hypothesized that the lipid identification algorithm may be sufficiently specific also if larger compositional variations are allowed. In this way, we might be able to identify lipids composed of similar “building blocks”, yet the composition of each block may substantially differ from the source structure. To test the identification specificity, we spiked synthetic “clicked-PC” (cPC) lipids22 carrying the hydroxycoumaryltriazol group at the ω-position of a 18:1-fatty acid moiety (Figure 1B) into total lipid extracts from S. cerevisiae. In these experiments, cPC served as a positive control since they do not occur in yeast; also, the hydroxycoumaryl moiety gives rise to fragments absent in MS/MS spectra of native PC. At the same time, the cPC structure is generally similar to native PC (Figure 1B). Effectively, we tested the hypothesis if we could specifically detect PC variant species by screening a total lipid extract for a core structure with undefined modifications. In the shotgun acquisition of the yeast lipid extract (Figure 2), LipidXplorer recognized a total of 6900 peaks in MS spectra and 495 000 peaks in 600 MS/MS spectra acquired under a Q1 isolation window of 1 Th. To search for PC variants, we modified the above MFQL query by relaxing the compositional constraints:

Figure 2. (A) (−)FT MS spectrum of yeast lipid extract spiked with the clicked-PC; two major cPC species are indicated. (B) Relative abundances of cPC species reported by LipidXplorer. cFA stands for a moiety of the clicked fatty acid having the elemental composition of C28H36N3O5; another fatty acid moiety was unmodified. Black bars: yeast extract (cPC were not detected at all); dark gray: extracted cPC standard; light gray: yeast extract spiked with cPC. Because of background interference, low abundant cFA/18:1 species (designated with asterisk) was not directly identified in the spiked extracts but was recognized by retrospective inspection of the dump file created by LipidXplorer. Error bars indicate the standard deviation from duplicate extractions.

two atoms of phosphorus; this is also reflected in the definition of the composition of intact precursors in the variable PR. The SUCHTHAT section is basically the same as for native PC; for clarity here, we only show the query that assumes that lipids are detected as deprotonated molecules [M − H]− but not as acetate adducts as are native PC (a query targeting acetate adducts was also compiled and applied in a separate screen that returned no hits). Thus, the new query searched for phosphatidylcholine-like molecules in which one of the two fatty acids was modified in an undefined way and possibly included several heteroatoms. This definition of PR variable covered more than 31 000 chemically plausible formulas, and the total number of possible fragment combinations was close to 700 000. Expanding the range of elemental compositions increased the computation time to ca. 8.5 min compared to 18 s required for identifying native PC. The search result is shown in Figure 2B, where the relative abundances of identified species are compared between the spiked yeast extract, the extracted cPC sample itself, and the non-spiked yeast extract that served as negative control. In the

Instead of a variable FA1 (which stands for a carboxylate fragment) with a fixed number of heteroatoms (O[2]), the modified query now covers a range of heteroatom compositions: up to six atoms of nitrogen, eight atoms of oxygen, and 2706

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non-spiked extract, no cPC species were detected. Two major cPC species: cFA/16:0 and cFA/18:0 (where cFA stands for the clicked FA, Figure 1) were detected in the standard and in the spiked extract with identical relative abundances. The third minor cPC species cFA/18:1 was not reported as a bona f ide hit; however, it was found by retrospective inspection of the full list of retrieved lipid candidates. LipidXplorer correctly recognized its precursor in the MS spectrum and cFA carboxylate fragment in the corresponding MS/MS spectrum; however, the abundance ratio of cFA and 18:1 fragments was 4×-fold off the expected range because of strong background interference (Supporting Information Figure 2S and Table 1S). Importantly, no false positive hits (cPC species that were not detected in the standard itself) were reported by the analysis of spiked extracts. Hence, we concluded that the screening approach efficiently discriminated false positives; however, it might be missing some minor species because of possible interference of abundant background ions with very close precursor masses and producing a partially overlapping set of fragments, like carboxylate ions of yeast fatty acids. We note that, on user demand, LipidXplorer could output a dump file that reports all recognized precursor ions and specific fragments identified in the associated MS/MS spectra.21 If required, the dump file can be retrospectively inspected for other precursors that partially match the SUCHTHAT criteria. All species were correctly named according to the composition of fatty acid moieties; as expected, MS/MS was not able to pinpoint the structure of the modified moiety, yet its elemental composition was determined and reported correctly. We therefore concluded that novel variants of a known lipid class can be detected with high specificity from a crude lipid extract by only relying upon the assumed structural similarity and without the exact knowledge of the chemical modification and fragmentation pathways of the modified molecules. Screening for Maradolipid-Like Lipids in C. elegans. Maradolipids are 6,6′-di-O-acyltrehaloses that were recently discovered in the nematode C. elegans, where they exclusively occur in animals at the dauer stage,30 an arrested developmental variant produced in response to environmental stress. Their biological function is still poorly understood. Maradolipids are structurally related to diacyltrehaloses found in bacteria and fungi,31 most notably to a cord factor from M. tuberculosis.32 In other known fatty-acid-linked glycosides, the carbohydrate moieties may be modified by acetylation or sulfonation; also, they may carry a different number of fatty acid moieties33 that may also be hydroxylated. We wondered if other glycosides that are only distantly similar to maradolipids might also be present in C. elegans? To this end, we isolated polar lipid fractions by chromatography on a silica column of total extracts from animals at dauer and L3 larvae stages and analyzed them by shotgun mass spectrometry (Supporting Information Figure 3S). L3 larvae served as a representative stage of the reproductive life cycle of C. elegans. Both L3 and dauer larvae are produced at the second molt and have similar body sizes. While maradolipids 30 are not synthesized in L3 larvae, they were readily detectable in dauer extracts (Supporting Information Figure 3S). Upon HCD fragmentation, anions of acetate adducts of maradolipids produced carboxylate fragments of their fatty acid moieties and fragments originating from their neutral loss, as well as fragments related to the trehalose moiety30 (Figure 3A).

Figure 3. (A) (−)FT MS/MS spectrum of the acetate adduct of maradolipid standard 18:1/15:0. [R1COO]− and [R2COO]− indicate the carboxylate ions of the 15:0 and the 18:1 fatty acid moieties, respectively. (B) (−)FT MS/MS spectrum of the endogenous 15:0 lyso-maradolipid species shown in the inset. The ester bond cleaved by CID is indicated. (C) Relative abundances of fatty acid moieties in maradolipid (dark gray) and lyso-maradolipid species (light gray) as reported by LipidXplorer. The error bars indicate the standard deviation from duplicate independent extractions.

We used these fragmentation pathways (Supporting Information Figure 4S) as a template to design a series of MFQL queries that covered a large number of maradolipid variants. It was not possible to compact them into a single comprehensive “super”-query because MFQL does not support boolean OR-operation in the IDENTIFY section. The first query encompassed putative maradolipid variants that have the same “building blocks” composition as native maradolipids: they should contain a carbohydrate (TREH) and two fatty acid (FA1 and FA2) moieties; however, we assumed that TREH might bear additional chemical groups with multiple heteroatoms. This would account for common modifications such as acetylation, methylation, phosphorylation, sulfation, or any combination of those. The query also allowed up to one hydroxyl group attached to the hydrocarbon 2707

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unknown class of monoacyl-glycosides enriched in C. elegans dauers.

chains of FA1 and FA2. The SUCHTHAT section required that FA1, FA2, and TREH should complement each other to a full mass of the acetate adduct (note that in the maradolipids spectra the fragment TREH differs from the intact trehalose by loss of two water molecules).

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DISCUSSION This work rests on two key assumptions: first, in shotgun analyses, lipids are detected and fragmented, irrespective whether they belong to known or currently unknown lipid classes. By virtue of data acquisition alone, a shotgun data set should be complete: in principle, within hundreds of convoluted MS/MS spectra, it should contain all fragment ions produced from all precursors ionized under the given experimental conditions; although in practice, the detection of lipid species is limited by the instruments sensitivity and dynamic range, the compositional complexity of the analyte, and the ionization capacity of the ion source. It therefore only covers a fraction of the full lipidome. Second, we assumed that even unknown lipids may possess substantial compositional similarity to known lipid classes. Sharing common “building blocks” might translate into a commonality of their fragmentation pathways that, in turn, allows recognition of the possible species by the algorithm implemented in LipidXplorer software. We note that LipidXplorer does not attempt to interpret MS/MS spectra de novo or compare them to known resource spectra. Instead, it identifies combinations of anticipated key fragments that point to a plausible structure. It is not necessary to exactly define masses or compositions of anticipated fragments: this work proved that defining them as broad ranges is sufficiently specific and, at the same time, it drastically simplifies and accelerates screening. Although a single query encompassed hundreds of thousands of individual plausible formulas, no random (false positive) identifications were reported. Although the approach benefits from the high mass resolution and accuracy of modern tandem mass spectrometers, screening might not produce unequivocal identifications of new structures. It may only identify candidate precursors and their “building block” compositions. However, the exact structural assignment will require further in-depth investigations by mass spectrometry (including MSn experiments37), directed fragmentation methods such as real time ozonolyis,38 or even largescale biochemical isolation and NMR.39 Most lipid classes occur as series of molecules with identical heteroatom composition but varying in the chain length and unsaturation of hydrocarbon moieties. The identification of homologous members of the lipid class is covered by a single MFQL query and is important supporting evidence for distinguishing new lipid classes. A complete shotgun spectra data set could be subjected to retrospective interrogation for the fragmentation signatures of any real or putative lipid class. In principle, MFQL-driven screening might circumvent the need for repeating the biochemical isolation and mass spectrometric analysis for probing the lipidome for previously unanticipated components. Considering the nM-range sensitivity and over 10 000-fold dynamic range of modern high resolution tandem mass spectrometers,17 it is conceivable that a single analysis of a total lipid extract may reveal all major components while repetitive runs will be required for identifying relatively minor (not to say unimportant) lipids enriched by targeted biochemical purifications. The discovery of maradolipoids in C. elegans30 was a highly surprising finding: previously, acyltrehaloses were only isolated from unicellular organisms. Here, we investigated if maradoli-

The screen found no maradolipid-related molecules except maradolipids themselves, which in this case effectively served as a positive control. Since glycosides with anionic groups (like phosphates or sulfates)34,35 may be detectable as deprotonated molecules [M − H]− rather than as acetate adducts, we modified the SUCHTHAT section to: We also enhanced the production of anions by adding triethylamine into the analyte. However, the search still produced no hits. Finally, we relaxed the requirement for having exactly two fatty acid moieties, while still allowing a large compositional variability of the carbohydrate core and optional hydroxylation of fatty acid moieties. Additionally, we compiled two separate queries allowing for one and for three fatty acid moieties (the variant with two moieties was considered above, and the search only returned maradolipids). Altogether, the queries covered more than 650 000 partially redundant putative structures.

This screen reported a series of monoacyl-maradolipid (lysomaradolipids) species that were similar to the Emmyguyacins A and B previously described in a sterile fungus species,36 except that in lyso-maradolipids the fatty acid moiety was unmodified. The structure of lyso-maradolipids was confirmed by MS/MS: in the spectra, we observed carboxylate fragments corresponding to the fatty acid moieties and the fragment of unmodified trehalose (Figure 3B). Quantitative interpretation of MS/MS spectra suggested that the distribution of relative abundances of fatty acids in lyso-mardolipids and maradolipids were different: maradolipids were enriched in 18:1 fatty acid, whereas in lysospecies 15:0, 17:0, and 18:1 moieties had almost equal abundance. Intriguingly, 18:0 was abundant in lyso-species, while it was almost absent in maradolipids (Figure 3C and ref 30). This suggested that lyso-species were not produced by random degradation of diacyl maradolipids during biochemical isolation or mass spectrometric analysis. Both lyso-maradolipids and maradolipids were specific for the dauer stage larvae. Taken together, screening the shotgun data set with four MFQL queries encompassed more than 650 000 putative structures and identified lyso-maradolipids as a single and previously 2708

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pids are the only dauer acylglycosides or whether other lipids structurally similar to known acylglycosides from bacteria and fungi could have been present, yet remained unidentified because of their low abundance and paucity of analytical methodologies. While the identification of lyso-maradolipids is important and prompts further investigation of their biological role and relationship to maradolipids, it is equally important that this screen ruled out alternative forms whose abundance might be comparable with these species.

CONCLUSIONS AND PERSPECTIVES This work serves as a proof-of-concept study for the identification of species of unknown lipid classes by structuresimilarity driven shotgun lipidomics. Despite the fact that MFQL queries did not consider the exact compositions of expected fragments and did not rely upon the exact knowledge of the fragmentation mechanisms, in the control experiments with total lipid extracts, searches produced no random hits and correctly identified the target molecules. In a real-world test, the screen recognized a previously unknown class of lysomaradolipids that was specifically enriched in C. elegans at the dauer stage and, at the same time, ruled out the occurrence of multiple alternative forms of acylglycosides. Even in its current implementation, structure-similarity driven lipidomics is a workable analytical method. However, several developments may further enhance its efficiency. Because of the high (and steadily increasing) spectra acquisition rate of tandem mass spectrometers, it is important to develop better data acquisition routines that may yield several complementary MS/MS spectra for each fragmented precursor: acquisitions may utilize different fragmentation mechanisms such as CID or higher-energy collisional dissociation (HCD); MSn; and/or varying collision energies. Also, it should be possible to automate the design of MFQL queries to support screening for variants of core structures using software with a simple intuitive graphical interface. Finally, since the approach supports retrospective screening for any assumed variant structure, a repository of complete shotgun spectra data sets, acquired from extracts of species whose lipidomes are currently insufficiently characterized, may become a valuable resource for the entire lipidomics community. ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Work in AS and CT laboratories was supported by TRR 83 grant from Deutsche Forschungsgemeinschaft (DFG) (projects A17 and A12, respectively) and Virtual Liver Network grant (Code/0315757) from Bundesministerium f. Bildung u. Forschung (BMBF). We thank Prof. H.-J. Knölker (Technical University of Dresden) for providing synthetic maradolipid standards. 2709

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