Published: June 02, 2011

r 2011 American Chemical Society 5480 dx.doi.org/10.1021/ac102505f |Anal. Chem. 2011, 83, 5480–5487

ARTICLE

pubs.acs.org/ac

Bottom-Up Shotgun Lipidomics by Higher Energy CollisionalDissociation on LTQ Orbitrap Mass SpectrometersKai Schuhmann,†,‡ Ronny Herzog,†,‡ Dominik Schwudke,†,§ Wolfgang Metelmann-Strupat,||

Stefan R. Bornstein,‡ and Andrej Shevchenko†,*†Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany‡Department of Internal Medicine III, Technical University of Dresden, 01307 Dresden, Germany§National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK, Bellary Road, Bangalore 560065, India

)Thermo Fisher Scientific GmbH, Im Steingrund 4-6, 63303 Dreieich, Germany

bS Supporting Information

Lipidomics is a branch of omics sciences that aims atquantifying a full complement of lipid molecules in cells,

tissues, or organisms (reviewed in refs 1�4). According todifferent estimates, eukaryotic lipidomes might comprise10 000 to 100 000 individual species originating from a fewhundred lipid classes.5�7 Developments in mass spectrometry(reviewed in refs 8 and 9) and analytical methods forgedlipidomics into a recognized scientific discipline (reviewed inref 10). It encompasses all major lipid classes, includingglycolipids11�13 and enables quantifying individual molecularspecies at the lipidome-wide scale (reviewed in refs 14 and 15).

Bottom-up lipidomics relies on tandem mass spectrometricexperiments that are either performed on-line with liquidchromatography or by infusion of total lipid extracts directlyinto a spectrometer. The latter approach is also termed shotgunlipidomics,16 by analogy to much used shotgun genomicsequencing. It is generally assumed that collision-induceddissociation of lipid precursors produces fragment ions thatare either characteristic for the entire lipid class or distinguishspecific structural moieties in individual species. Principles ofselecting specific fragment ions and scanning modes are exten-sively reviewed in refs 10,16�18. Initially shotgun lipidomicsrelied on successive precursor and neutral loss scans (reviewedin ref 16). Lately, with the use of hybrid tandem mass spectro-meters and a robotic nanoflow ion source, it has becomepossible to acquire full tandem mass spectra from hundreds ofplausible lipid precursors.19 Their automated post-acquisitioninterpretation effectively emulates an unlimited number ofprecursor and neutral loss scans and also bundles them by logical

operations.19�21 Shotgun profiling by data-dependent acquisi-tion was first developed for and applied with hybrid quadrupoletime-of-flight mass spectrometers. Both fatty acid scanning22

and data-dependent MS/MS experiments19 consistently quan-tified molecular species of a variety of lipid classes.11,23�26

Hybrid linear ion trap�Orbitrap tandemmass spectrometers27,28

have developed into an important lipidomics tool because ofrapid acquisition of MS/MS spectra, higher mass resolution, andoptional MSn fragmentation.29,30 However, lipid fragmenta-tion pathways in ion traps and linear collision cells differsignificantly,30,31 and the yield of structurally informative acylanion fragments is often reduced. Furthermore, ion trap isolationat the unit or higher mass resolution might degrade unstableprecursors.32 It was therefore not uncommon to employ severalhigh-end tandem mass spectrometers within a lipidomics pipe-line. For example, high-resolution MS spectra could be acquiredat a LTQ Orbitrap, whereas MS/MS experiments were subse-quently performed on a QqTOF.11

Recently introduced LTQ Orbitrap Velos instruments33

equipped with a dual pressure linear ion trap supports high-resolution isolation of precursor ions.34 They could be furtherchanneled to and dissociated in a multipole collision cell and pro-duced fragments detected at the Orbitrap mass analyzer. Throughmultiple collisions of precursor and fragment ions with nitrogenmolecules, higher energy collision-induced dissociation (HCD)

Received: September 24, 2010Accepted: June 2, 2011

ABSTRACT: Higher energy collision dissociation (HCD) is a complementary frag-mentation tool that has recently become available on mass spectrometers of the LTQOrbitrap family. We report on a shotgun bottom-up lipidomics approach that relies onHCD of the isolated lipid precursors. HCD, together with the high mass resolution andmass accuracy of the Orbitrap analyzer, improved the confidence of molecular speciesassignment and accuracy of their quantification in total lipid extracts. These capabilitieswere particularly important for accounting for biologically interesting lipid speciescomprising polyunsaturated and odd numbered fatty acid moieties. We argue that nowboth bottom-up and top-down shotgun lipidomics could be performed on the sameinstrumentation platform.

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enhanced the yield of stable low molecular weight fragments.33

Note that HCD does not imply high (in the range of hundreds ofelectronvolts) collision energies. Similar to conventional triplequadrupole or quadrupole time-of-flight machines with linearcollision cells, a typical collision energy in HCD experiments ismaintained within the range of 10�100 eV. Highmass resolutionandmass accuracy of theOrbitrap helps in distinguishing isobaricfragments and their unequivocal assignment to individual spe-cies, even if several precursor ions with closem/zwere coisolatedfor MS/MS experiments.

We set out to establish a bottom-up shotgun lipidomicsroutine that relies on the characterization of individual molecularspecies by HCD. We demonstrated that HCD, together withbetter than 100 000 mass resolution of the Orbitrap, improvedthe confidence of molecular species assignment and accuracy oftheir quantification, which was particularly important for lowabundant, yet biologically important, species comprising poly-unsaturated and odd numbered fatty acid moieties. The remark-able performance of LTQ Orbitrap instruments in bottom-uplipidomics has lead us to the notion that now both bottom-upand top-down analysis could be run on the same instrumentationplatform.

’MATERIALS AND METHODS

Chemicals and Lipid Standards. Synthetic lipid standardsand a total lipid extract of bovine heart were purchased fromAvanti Polar Lipids, Inc. (Alabaster, AL) or Sigma-AldrichChemie (Munich, Germany); common chemicals and solventsof ACS or LC�MS grade from Sigma-Aldrich Chemie (Munich,Germany) or Fluka (Buchs SG, Switzerland);methanol (LiChrosolvgrade) from Merck (Darmstadt, Germany).Annotation of Lipid Species. Lipid classes: PE, phosphati-

dylethanolamines; PE-O, 1-O-alkyl-2-acylglycerophosphoetha-nolamines; LPE; lyso-phosphatidylethanolamines; PS, phos-phatidylserines; PC, phosphatidylcholines; PC-O, 1-O-alkyl-2-acylglycerophosphocholines; LPC, lysophosphatidylcholines;SM, sphingomyelins; PI, phosphatidylinositols; TAG, triacylglycer-ols; Cer, ceramides; CL, cardiolipins; GlcCer, glucosylceramides.Individual molecular species were annotated as follows:

Ælipid classæ Æno. of carbon atoms in the first fatty acid or fattyalcohol moietyæ:Æno. of double bonds in the first fatty acid orfatty alcohol moietyæ/Æno. of carbon atoms in the second fattyacid moietyæ:Æno. of double bonds in the second fatty acidmoietyæ. For example, PC 18:0/18:1 stands for a phosphati-dylcholine comprising the moieties of stearic (18:0) and oleic(18:1) fatty acids. If the exact composition of fatty acid or fattyalcohol moieties was unknown, the species were annotated asÆlipid classæ Æno. of carbon atoms in both moietiesæ:Æno. ofdouble bonds in both moietiesæ. In this way, PC 36:1 stands forthe PC species having 36 carbon atoms and one double bond inboth fatty acid moieties.Total lipid extract from rat retina was prepared as described35

with minor modifications. Briefly, a sample of rat retina tissue(wet weight ∼7 mg) was homogenized using a Dounce tissuegrinder (Sigma-Aldrich Chemie, Munich) and extracted with700 μL of methyl-tert-butyl ether (MTBE)/methanol 10:3 (v/v).Synthetic standards of PC 17:0/17:0; PE 17:0/17:0; PS 12:0/12:0, and PI 8:0/8:0 were spiked into theMTBE/MeOHmixtureprior to lipid extraction in the concentration of 10 μM each andthe mixture was incubated at 4 �C for 1 h on a shaking platform.To initiate phase separation, 135 μL of water were added and the

mixture was shaken for another 15 min. Phase separation wascompleted upon centrifuging for another 5 min in a benchtopcentrifuge MiniSpin (Eppendorf, Hamburg) at 13 400 rpm. Theupper organic phase was collected and stored at �25 �C undernitrogen.Samples forMass Spectrometric Analysis.Retina total lipid

extracts or lipid standards were diluted with a mixture ofisopropanol/methanol/chloroform 4:2:1 (v/v/v) containing7.5 mM ammonium formate or ammonium acetate, as indicatedseparately for each experiment. Also, where specified, we used amixture of methanol/chloroform 5:1 (v/v) containing 0.1%(v/v) triethylamine. Prior to the analysis, samples were loadedinto a 96-well plate (Eppendorf, Hamburg), sealed with alumi-num foil, and centrifuged for 5 min at 4 000 rpm on a Multifuge3S-R centrifuge from Heraeus DJB Labcare Ltd. (NewportPagnell, U.K.). The retina extract was diluted 10 times prior tothe analysis. Final concentrations of synthetic standards wereselected individually for each experiment.Mass spectrometric analses were performed on the LTQ Orbi-

trap XL (further termed as XL) and LTQOrbitrap Velos (furthertermed asVelos) instruments (ThermoFisher Scientific, Bremen),both equipped with a robotic nanoflow ion source TriVersa(AdvionBioSciences, IthacaNY) using chips with spraying nozzleswith a diameter of 4.1 μm. The ion source was controlled byChipsoft 6.4 software. The ionization voltage and gas backpressurewere set to 1.25 kV and 0.95 psi in the positive and 0.7 kV and 1.06psi in negative ionmodes, respectively. Under these settings, 10μLof the analyte was electrosprayed for more than 30 min.The temperatures of the ion transfer capillary were 125 and

200 �C for the XL and Velos, respectively; the tube voltages were90 V (MSþ) or �150 V (MS�) for both machines; and thes-lens level was 58% for the Velos.Isolation of Lipid Precursor Ions on XL and Velos Ma-

chines. The abundance of isolated precursors was monitoredby the method of total ion mapping. To this end, MS/MSspectra were acquired with the ion trap (IT) or (wherespecified) with the Orbitrap (FT) analyzer, while the targetprecursor m/z was changed with step increments of 0.1 Th.Precursor isolation width (1.0 Th); maximum injection time(100 ms), automated gain control (AGC) (5 000 ions), andnormalized collision energy (nCE) (1%) were fixed, while thetarget precursor m/z was altered in 0.1 Th step increments.Optimal offset values for species of different lipid classes were

determined by analyzing in replicate in the negative ion mode amixture of synthetic lipid standards LPC 12:0, PC 12:0/12:0, PC14:0/14:0, PC 22:0/22:0, PC 24:0/24:0, and Cer d18:1/17:0;CL 14:0/14:0/14:0/14:0, GlcCer d18:1/17:0, PG 17:0/17:0,SM d18:1/17:0, and PC 17:0/17:0, each at a concentration of2.5 μM. Total ion maps were acquired under the followingsettings: nCE was 1%; isolation width was 1.5 Th; ITmax was 250ms; target value for AGC was 5 000; and target mass resolutionat m/z 400 (Rm/z400) was 7 500 (full width at half maximum,FWHM).Lipid fragmentation in collision induced dissociation (CID),

pulsed Q collision induced dissociation (PQD), and HCDmodes was compared by acquiring collision energy profiles forthe synthetic lipid standard PE 18:1/18:2. The analyte with aconcentration of 0.5 μMwas infused into a mass spectrometer inthe negative ion mode, and the abundances of precursor andfragment ions were plotted against the normalized collision energy.MS/MS spectra were acquired under the following commonsettings: isolation width was 1.5 Th; ITmax was 100 ms; AGC

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value was set to 25 000; target mass resolution Rm/z400 was30 000; for each spectrum, 3 scans were averaged within the totalacquisition time of 1.19 s.Identification and Quantification of Molecular Species in

Total Lipid Extracts. Centroided HCD FT MS/MS spectrawere acquired in data-dependent mode as described.19 Eachdata-dependent acquisition (DDA) cycle consisted of one FTMS survey spectrum acquired at the target resolution Rm/z400 of100 000, followed by the acquisition of five HCD FT MS/MSspectra at the resolution Rm/z400 of 30 000. The prescan optionwas disabled. Precursor ions were subjected to MS/MS if theirm/z matched the masses in the inclusion list with the accuracyof (5 ppm. In MS/MS experiments, precursor ions wereisolated at the linear ion trap with the isolation width of 1.6Th, and the ITmax was set to 4 s. Exclusion time was set to20 min. On average, a DDA experiment was completed in40 min such that each precursor was fragmented twice. In HCDmode, nCE was 45%; fragments (m/z g 100) were detectedby the Orbitrap analyzer at the resolution Rm/z400 of 30 000.The maximum scan time for acquiring one HCD FT MS/MSspectrum was 5.4 s. The lock mass option was enabled andabundant background anion of octadecyl-(di-tert-butyl-hydro-xyphenyl)propionate ([M � H]�) with m/z 529.46262 wasused as a reference peak. Target AGC values were set to 1� 106

and 2.5 � 104 for FT MS and FT MS/MS modes, respectively.In FTMS, ITmax was 100 ms and 3 scans were averaged for eachspectrum. DDA experiments were repeated four times for eachsample and twice for the blank.Lipid species were identified by LipidXplorer software as

described in the Supporting Information, part 1. Lipid specieswere quantified by comparing the abundances of precursor peaksin high-resolution MS spectra and acyl anions peaks in MS/MSspectra with corresponding peaks of precursors and fragments ofspiked internal standards.19,20 For quantifying rat retina lipids,abundances of monoisotopic peaks were adjusted according toisotopic profiles calculated from elemental compositions ofcorresponding molecular ions to compensate the large differencein the length of fatty acid moieties.LipidXplorer installer, MFQL libraries, and a lipid identifica-

tion tutorial are available through https://wiki.mpi-cbg.de/wiki/lipidx/index.php/Main_Page

’RESULTS AND DISCUSSION

Accurate Isolation of Lipid Precursor Ions by the LinearIon Trap. If lipid species of the same class differ by a double bondin their hydrocarbon moieties, their precursors are spaced by am/z difference of 2 Th. Since in MS/MS experiments theseprecursors may produce the same specific fragment (like, thesame headgroup), it would be advantageous to fragment theirpeaks separately, and therefore in data-dependent shotgunexperiments precursors are typically isolated at the unit or highermass resolution.19

To determine the abundance profile of precursor ions isolatedat the unit resolution of the linear trap, we acquired MS/MSspectra of PC 16:0/22:6 in positive and negative ion modes withthe XL and Velos. Normalized collision energy was maintained at1% such that precursors remained intact. Note that in theseexperiments we could not employ internal standards to com-pensate for spraying instability, which typically contributed toless than 12% variation of the lipid peaks abundance (SupportingInformation part 2, Figure S13).

To maximize the abundance of isolated precursors, a massoffset (the difference between the precursor m/z and m/ztargeted in MS/MS experiment) should be applied on both theXL and Velos machines (Figure 1 and Supporting Information,part 2, Table S1). The unequal offset applied at XL and Velosmachines could be explained by the difference in design of lineartraps and ion isolation procedures.34

Further experiments with lipid standards suggested that atboth XL and Velos, the mass offset did not change if AGCwas set

Figure 1. Relative abundance of the precursor ion of PC 16:0/22:6isolated by the linear ion trap at the XL and Velos in (A) positive mode([MþH]þ; calculated m/z 806.5694) and (B) negative mode ([MþHCOO]�; calculated m/z 850.5593). In positive mode, the averageprecursor m/z measured in MS experiments were 806.42 (�0.15 Th)on IT; 806.5684 (�1.24 ppm) on Orbitrap (FT) at the XL; and 806.65(þ0.08 Th) on IT at the Velos. In negative mode, the averageprecursor m/z measured in MS experiments were 850.32 (�0.24Th) on IT; 806.5603 (�0.10 ppm) on Orbitrap (FT) at the XL; and850.5609 (0.62 ppm) on FT at the Velos. At the x-axis, the mass offsetwas calculated as the difference between the target and calculatedprecursorm/z and was not adjusted for the mass calibration; y-axis, theprecursor ion abundance was normalized to the maximum valueobserved in each experiment. IT/IT stands for theMS/MS experimentin which the precursor ion was isolated and detected at the ion trap;IT/FT, precursor was isolated at the ion trap and detected at theOrbitrap.

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to the higher target value (30 000 instead of 5 000 used toproduce data for Figure 1). At the same instrument, the offsetdid not change between positive and negative modes and showedday-to-day variation within the range of 0.1 Th, which corre-sponded to onem/z step at the total ion map plot (Figure 1). Forthe same polarity, the offset was also unaffected by changing theisolation width within 1.0 to 1.8 Th, although the abundance ofisolated peaks changed considerably (Supporting Information,part 2, Figure S1).We were also concerned if offsets would be substance

dependent since isolation under the unit resolution mightdegrade unstable precursors.36,37 Therefore, we further ac-quired total ion maps from precursors of 11 synthetic lipidstandards and determined optimal offset values and theirvariations (Figure 2). With the isolation width of 1.5 Th, theoptimal offset was, on average, 0.4 Th independently of the lipidclass or m/z of the species and was within the error margin setby varying ion current and spraying stability.We therefore concluded that applying mass offsets is im-

portant for the unit isolation of lipid precursors. The actualoffset values depend on the ion trap design but are weaklyaffected by acquisition polarity, AGC target values, width of theisolation window, as well as m/z and chemical structures ofisolated lipid precursor. Once determined in experiments withreference substances, the same offset could be applied forprofiling of multiple lipid classes.How isolation width affects the absolute abundance of pre-

cursor ions? Lipids are usually detected as molecular cations oranions but also as adducts with chloride or acetate anions innegative or lithium or ammonium cations in positive ion mode,respectively (reviewed in refs 8 and 10), which differ by theircollisional stability. Therefore we fixed the mass offset and nCEand determined how the abundance of monoisotopic peaks wasaffected by the isolation width.For comparative testing we used PE 18:0/22:6 detected as

[M � H]� and PC 16:0/22:6 detected as [M þ AcO]� in thenegative ion mode (Figure 3).

The monoisotopic peak of the molecular anion of PE could beisolated with no major abundance losses down to the width of1.0 Th. In contrast, under the same settings the monoisotopicpeak of the acetate adduct of PC was undetectable, while itsintensity gradually increased with increasing the isolation width.Full MS/MS spectra contained no [M � 15]� fragment ions ofdemethylated PC.31 We reasoned that unit resolution isolationrapidly degraded the [M þ AcO]� precursor, while its majorcollisional dissociation product [M � 15]� was not trapped.Although we could detect the peak of acetate adduct at the Veloseven at the 1.0 Th isolation window, its abundance was 2.5-foldlower compared to the isolation at the “fully opened” window of2.0 Th.We observed that molecular ions of several lipid classes, such

as sphingolipids, suffered even larger abundance losses. It was

Figure 2. Optimal offset value is independent of the lipid class and/orm/z of lipid species. Total ion maps were acquired from precursor ionsof 11 synthetic lipid standards (names boxed) at nCE of 1% and isolationwidth of 1.5 Th. Optimal offset values correspond to the maximalabundance of isolated monoisotopic peaks. Measurements were per-formed in triplicate in negative ion mode on an XL machine.

Figure 3. Abundance of isolated monoisotopic peaks depends on pre-cursor ion stability. (A) Isolation of [M þ AcO]� precursor ion of PC16:0/22:6 detected by FT MS/MS at m/z 864.5740 (þ1.73 ppm); targetm/z 865.0. (B) Isolation of [M � H]� precursor ion of PE 18:0/22:6detected by FTMS/MS atm/z 790.5392 (þ1.39 ppm); targetm/z 790.8.Experiments were performed on an LTQ Orbitrap XL instrument with anCE of 1%. Intensities of the first isotopic peaks are provided as references;however, peaks were only observed if the isolation window exceeded 2 Th(see the Supporting Information, part 2, Figure S2). Error bars werecalculated on the basis of three independent experiments while no referencesignals compensating for the ion current instability were employed.

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almost impossible to isolate their [M þ H]þ precursors withinthe width of less than 3 Th and their collisional instability

(“fragility”, according to refs 36 and 37) remains a major bottle-neck of bottom-up shotgun lipidomics experiments on iontrap instruments. In our experience, it could be alleviated bytargeting different molecular ion forms, applying intrasourceprecursor ion separation38 and isolating ions at the broaderwidth.HCD as a Resource for Lipid Species Profiling. Once

precursor ions have been isolated in the ion trap, both theXL and Velos offer multiple ways for their subsequent frag-mentation. They can be fragmented in the linear ion trap inthe CID or PQD modes or in the HCD mode in the sepa-rate multipole collision cell attached to the C-trap. Herewe examined the analytical merits of different fragmentationmeans for identifying molecular species of common glycero-phospholipid classes.Molecular species of glycerophospholipids are usually identi-

fied by their accurate precursor masses and acyl anion fragmentsof their fatty acid moieties. Using the standard of PE 18:0/18:1,we compared the efficiency of precursor ion fragmentation andthe yield of acyl anions in CID, PQD, and HCD modes(Figure 4).Although the precursor ion could be completely fragmented

by all three methods, we see two reasons why the most abundantacyl anion fragments were produced by HCD. First, HCD is amultiple collision process. Similar to the fragmentation inclassical triple quadrupole instruments, higher CE drives thedegradation of both precursors and intermediates down tothe most stable acyl anion fragments. Second, the HCD cell�C-trap�Orbitrap scheme is more permissive toward transmis-sion and detection of low-molecular weight fragments, comparedto conventional ion traps:39 it is only limited by rf-amplitude ofthe C-trap and cuts off m/z below (approximately) 1/20 of theprecursorm/z and therefore does not compromise the detectionof acyl anion fragments.Quantitative profiling of glycerophospholipid species via acyl

anions of their fatty acid moieties is an established analyticalapproach used on hybrid quadrupole time-of-flight mass spectro-meters in multiple precursor ion scanning (MPIS)11,40 and data-dependent acquisition (DDA)19 modes. Quantitative profilesobtained in independent DDA and MPIS experiments corrobo-rate,19 and the linear dynamic range of species detection exceeds10 000-fold.11,40 Here we examined if HCD of glycerophospho-lipid anions could be applied for molecular species profiling inthe same way.We observed that in HCD mode, normalizing the collision

energy offset enabled equally efficient fragmentation of the sameclass species, irrespective of their m/z (Supporting Information,part 2, Figure S3). Then we tested how the yield of acyl anionsdepended on the lipid class and nCE (Figure 5).Similar to collision energy profiles typical for QqTOF

instruments,40 HCD of PI required slightly higher nCE com-pared to PC and PE, while maximum intensity for the acyl anionsof all species was achieved at nCE of 65%.We then estimated the dynamic range of HCD quantifica-

tion of lipids of different classes. Test samples were obtainedby serial dilution of a mixture of seven synthetic lipid stan-dards. Precursor masses were subjected to HCD FT MS/MS,and the combined intensity of peaks of acyl anions plottedagainst the species concentrations. We observed the linearresponse within better than a 1000-fold concentration rangefor each of the quantified standards (Supporting Information,part 2, Figure S5).

Figure 4. Normalized collision energy profiles of PE 18:0/18:1 (m/z744.5549) produced by CID (panel A), PQD (panel B), and HCD(panel C) acquired on the Velos machine. m/z of fragment ions in theinset: m/z 283.2643, acyl anion of 18:0 fatty acid; m/z 281.2486, acylanion of 18:1 fatty acid. NLs stands for the combined intensity of m/z480.3091 andm/z 478.2933, the products of neutral loss of ketens of 18:1and 18:0 fatty acids, respectively. TIC stands for the total ion current.

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We therefore concluded that, on instruments of the LTQOrbitrap family, HCD outperformed CID and PQD in quanti-tative molecular species profiling. Normalization of the collisionenergy enabled one to achieve an equally complete yield of acylanions from glycerophospholipid precursors independent oftheir m/z, molecular forms (molecular anions or anion adducts),and class.HCD and FT MS/MS for Accurate Quantification of Lipids

with PUFA Moieties. HCD, together with high mass resolution

and part per million mass accuracy enabled unequivocal assign-ment of fragment ions in MS/MS spectra and was particularlyimportant for the quantification of lipids with polyunsaturatedfatty acids (PUFA) moieties. In MS/MS, acyl anion fragments ofPUFA species undergo further fragmentation with neutral loss ofCO2.

41,42 In this way, acyl anion of docosahexaenoic (22:6) fattyacid produces a fragment with calculated m/z 283.2431, which isisobaric to the acyl anion of abundant stearic (18:0) fatty acid(m/z 283.2643) (see the Supporting Information, part 2, FigureS6 for chemical structures). While their peaks overlap in low-resolution MS/MS spectra, they were completely resolved in FTMS/MS spectra (Figure 6) and therefore accurate quantificationof the PUFA species did not rely upon arbitrary corrections of peakabundances,40 yet required careful selection of nCE (see below).Glycerophospholipidome of Rat Retina by HCD FTMS/MS.

We appliedHCD to profile a total lipid extract of rat retina, whichis enriched in glycerophospholipid species with PUFAmoieties.43

The analysis of, in total, 7 mg of tissue was performed in twobiological replicas, each of which was analyzed in four technicalreplicas. Each shotgun analysis consumed 820 pmol of thetotal lipid material and produced a data set of 125 FT MS and600 HCD FT MS/MS spectra acquired using the inclusion listof precursorm/z computed from expected elemental compositionsof glycerophospholipid species.19,20 Because of the isolationwidthof 1.5 Th, isobaric precursors were fragmented together, whileindividual species were recognized and quantified in “mixed”MS/MS spectra by lipid class-specific and lipids species-specificfragment ions. Molecular species (Figure 7) were identified byLipidXplorer software (see the Supporting Information, part 1 fordetails).Bulk lipid class composition independently determined by

HCD FT MS/MS and by FT MS in positive and negativemodes (see the Supporting Information, part 2, Figure S8)corroborated previous publications on mammalian retinas.44,45

To the best of our knowledge, this is the first report on theabundance of individual molecular species, including specieswith very long chain (VLC) PUFA moieties.45 We underscorethat, because of its focus on glycerophospholipidome, this

Figure 6. HCD FTMS/MS of PE 18:0/22:6 ([M�H]�) acquired at Rm/z400 of 30 000 on the XLmachine. Peaks of the acyl anion of FA 18:0 and theproduct of CO2 loss from the acyl anion of FA 22:6 were fully resolved (inset). Chemical structures of fragment ions are presented in the SupportingInformation, part 2, Figure S6. [FA 18:0]� and [FA 22:6]� designate acyl anion fragments of corresponding fatty acid moieties.

Figure 5. The abundance of acyl anion fragments of 16:0, 18:0, and 18:2fatty acid moieties produced by HCD of molecular anions of LPE 18:0(m/z 480.3096), PI 36:2 (m/z 861.5499), and PE 36:2 (m/z 742.5392)and of acetate adducts of PC 34:2 (m/z 816.5760) and PC-O 34:3(m/z 800.5800) that were fragmented during shotgun analysis of a bovineheart extract on the XL instrument. The abundance of each fragment ionwas normalized to its maximal value and represents the average of twoindependent experiments. While similar profiles were observed for otheracyl anion fragments from different species, only 16:0, 18:0, and 18:2 aredisplayed here for presentation clarity. Collision energy profiles of majorfragment ions of PC 34:2 and PE 36:2 are presented in the SupportingInformation, part 2, Figure S4.

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analysis did not encompass other important lipid classes, suchas sphingolipids or acylglycerols.Interestingly, we detected VLC PUFA species with 26�34

carbon atoms and up to 6 double bonds in which both fatty acidmoieties were highly unsaturated. As a representative example,HCD FT MS/MS spectrum of PC 32:6/22:6 is presented inSupporting Information, part 2, Figure S9. Despite that they areuncommon and relatively low abundant, their HCD identifica-tion was confident since it relied upon multiple fragmentsmatched with sub-ppm accuracy.Since exact quantities of individual species in rat retina were

unknown, we seek indirect means for validating that our quanti-tative assignments were correct. Lipid quantification methodrelying upon HCD FT MS/MS is conceptually similar to shot-gun methodology established on a quadrupole time-of-flightmass spectrometer.19,20,40 While adapting it to another instru-ment, we checked if the combined abundances of acyl anions andtheir neutral loss products in MS/MS spectra independentlycorroborated the abundances of intact precursors in MS spectrafor each lipid class. Indeed, we found that for the molecularspecies of four major classes (PC, PE, PI, and PS) averagecorrelation coefficient r2 between the quantitative estimatesbased on FT MS and HCD FT MS/MS spectra was of the valueof 0.992 (Supporting Information, part 2, Figure S10).High-resolution HCD FT MS/MS distinguished isobaric frag-

ments and enabled direct quantificationof specieswith 22:6 fatty acidmoieties. To test if neutral loss products were identified correctly, wetemporarily disabled accounting for CO2 loss fragments (seeSupporting Information, part 1) and checked if the abundances ofmajor PC species were affected. As expected, upon correction forneutral losses the content of PC species without PUFA was un-changed, while the content of PC species with PUFA increased by∼10% (see the Supporting Information, part 2, Figure S11).This, however, did not imply that accounting for CO2 losses

from PUFA acyl anions enables setting nCE at some high

arbitrary values, and no instrument specific tuning of nCE wasrequired. At nCE > 50%we observed substantial uncompensatedlosses in the abundances of PUFA fragments, in contrast to acylanions having zero to two double bonds (see the SupportingInformation, part 2, Figure S12).Therefore, for accurate account-ing of the PUFA-containing species nCEmight be lower than theoptimal value (Figure 5), despite overall detection sensitivity isslightly reduced.High resolution and mass accuracy of HCD FT MS/MS

spectra were also instrumental in dissecting molecular composi-tion of other lipid classes (in particular, triacylglycerols) bydiscriminating ether species from isobaric species comprisingodd numbered fatty acid moieties (see the Supporting Informa-tion, part 2, Figure S13).Taken together, we demonstrated that HCD FT MS/MS

supported accurate quantitative profiling of molecular species ofa variety of lipid classes, including species comprising polyunsa-turated or odd numbered fatty acid moieties.

’CONCLUSIONS AND PERSPECTIVES

In this work we established HCD, a fragmentation methodrecently introduced on hybrid LTQ Orbitrap tandem massspectrometers, as a powerful tool for quantitative profiling ofmolecular species of glycerophospholipids. High mass resolutionand relative abundance of structurally specific acyl anion frag-ments were the two important factors that increased both theconfidence in molecular species assignment and quantifica-tion accuracy. We further demonstrated that LipidXplorer soft-ware took full advantage of the high mass resolution and massaccuracy in both MS and MS/MS spectra and supported theiraccurate and consistent interpretation. Therefore it could now bepossible to perform both top-down and bottom-up lipidomics ona single instrumentation platform and support both approachesby the same software.

Figure 7. Major molecular species of glycerophospholipids in rat retina. For presentation clarity, only 50 species whose abundances exceeded 75 pmol/mg are shown. The full list comprising 211 molecular species is provided in the Supporting Information, part 3. Note that negative ion mode HCD FTMS/MS did not distinguish ether lipids from plasmalogens.

5487 dx.doi.org/10.1021/ac102505f |Anal. Chem. 2011, 83, 5480–5487

Analytical Chemistry ARTICLE

This work also revealed important technical limitations of LTQOrbitrap instruments. Despite marked improvements in the iontrap technology, isolation of precursors with unit or higher massresolution was impacting unstable precursors. Although currentlyno generic solution is available, in many (yet, not all) instances itmight be possible to minimize it by using larger width of isolationwindows or analyzing alternative molecular forms (such as ad-ducts) with enhanced collisional stability. In summary, the highmass resolution of tandem mass spectrometers in both MS andMS/MSmodes, together with a palette of fragmentation methodsand software, now brings the potential to understand the sig-nificance of the compositional complexity of lipidomes for cellbiology and molecular medicine.14

’ASSOCIATED CONTENT

bS Supporting Information. Additional information asnoted in text. This material is available free of charge via theInternet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*E-mail: shevchenko@mpi-cbg.de.

’ACKNOWLEDGMENT

We are grateful for our colleagues at MPI CBG and theTechnical University of Dresden for collaboration and support.We are particularly thankful to Dr. Christer S. Ejsing (Universityof Southern Denmark, Odense) for his valuable input and usefuldiscussions and to Kathleen Eeisenhofer for critical reading of themanuscript. We are grateful for Dr.Ekaterina Lobanova an Prof.Vadim Arshavsky (Duke University Eye Center, Durham NC)for providing rat retina samples. Work in the AS laboratory wassupported by the TRR 83 Grant from Deutsche Forschungsge-meinschaft (DFG) and the Virtual Liver (Code/0315757) Grantfrom Bundesministerium f. Bildung u. Forschung (BMBF). D.S.is supported by the Wellcome Trust/DBT India Alliance.

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