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Bottom-Up Shotgun Lipidomics by Higher Energy Collisional Dissociation on LTQ Orbitrap Mass Spectrometers Kai 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 ABSTRACT: Higher energy collision dissociation (HCD) is a complementary fragmentation tool that has recently become available on mass spectrometers of the LTQ Orbitrap family. We report on a shotgun bottom-up lipidomics approach that relies on HCD of the isolated lipid precursors. HCD, together with the high mass resolution and mass accuracy of the Orbitrap analyzer, improved the confidence of molecular species assignment and accuracy of their quantification in total lipid extracts. These capabilities were particularly important for accounting for biologically interesting lipid species comprising polyunsaturated and odd numbered fatty acid moieties. We argue that now both bottom-up and top-down shotgun lipidomics could be performed on the same instrumentation platform.
L
ipidomics is a branch of omics sciences that aims at quantifying a full complement of lipid molecules in cells, tissues, or organisms (reviewed in refs 14). According to different estimates, eukaryotic lipidomes might comprise 10 000 to 100 000 individual species originating from a few hundred lipid classes.57 Developments in mass spectrometry (reviewed in refs 8 and 9) and analytical methods forged lipidomics into a recognized scientific discipline (reviewed in ref 10). It encompasses all major lipid classes, including glycolipids1113 and enables quantifying individual molecular species at the lipidome-wide scale (reviewed in refs 14 and 15). Bottom-up lipidomics relies on tandem mass spectrometric experiments that are either performed on-line with liquid chromatography or by infusion of total lipid extracts directly into a spectrometer. The latter approach is also termed shotgun lipidomics,16 by analogy to much used shotgun genomic sequencing. It is generally assumed that collision-induced dissociation of lipid precursors produces fragment ions that are either characteristic for the entire lipid class or distinguish specific structural moieties in individual species. Principles of selecting specific fragment ions and scanning modes are extensively reviewed in refs 10,1618. Initially shotgun lipidomics relied on successive precursor and neutral loss scans (reviewed in ref 16). Lately, with the use of hybrid tandem mass spectrometers and a robotic nanoflow ion source, it has become possible to acquire full tandem mass spectra from hundreds of plausible lipid precursors.19 Their automated post-acquisition interpretation effectively emulates an unlimited number of precursor and neutral loss scans and also bundles them by logical r 2011 American Chemical Society
operations.1921 Shotgun profiling by data-dependent acquisition was first developed for and applied with hybrid quadrupole time-of-flight mass spectrometers. Both fatty acid scanning22 and data-dependent MS/MS experiments19 consistently quantified molecular species of a variety of lipid classes.11,2326 Hybrid linear ion trapOrbitrap tandem mass spectrometers27,28 have developed into an important lipidomics tool because of rapid acquisition of MS/MS spectra, higher mass resolution, and optional MSn fragmentation.29,30 However, lipid fragmentation pathways in ion traps and linear collision cells differ significantly,30,31 and the yield of structurally informative acyl anion fragments is often reduced. Furthermore, ion trap isolation at the unit or higher mass resolution might degrade unstable precursors.32 It was therefore not uncommon to employ several high-end tandem mass spectrometers within a lipidomics pipeline. For example, high-resolution MS spectra could be acquired at a LTQ Orbitrap, whereas MS/MS experiments were subsequently performed on a QqTOF.11 Recently introduced LTQ Orbitrap Velos instruments33 equipped with a dual pressure linear ion trap supports highresolution isolation of precursor ions.34 They could be further channeled to and dissociated in a multipole collision cell and produced fragments detected at the Orbitrap mass analyzer. Through multiple collisions of precursor and fragment ions with nitrogen molecules, higher energy collision-induced dissociation (HCD) Received: September 24, 2010 Accepted: June 2, 2011 Published: June 02, 2011 5480
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Analytical Chemistry enhanced the yield of stable low molecular weight fragments.33 Note that HCD does not imply high (in the range of hundreds of electronvolts) collision energies. Similar to conventional triple quadrupole or quadrupole time-of-flight machines with linear collision cells, a typical collision energy in HCD experiments is maintained within the range of 10100 eV. High mass resolution and mass accuracy of the Orbitrap helps in distinguishing isobaric fragments and their unequivocal assignment to individual species, even if several precursor ions with close m/z were coisolated for MS/MS experiments. We set out to establish a bottom-up shotgun lipidomics routine that relies on the characterization of individual molecular species by HCD. We demonstrated that HCD, together with better than 100 000 mass resolution of the Orbitrap, improved the confidence of molecular species assignment and accuracy of their quantification, which was particularly important for low abundant, yet biologically important, species comprising polyunsaturated and odd numbered fatty acid moieties. The remarkable performance of LTQ Orbitrap instruments in bottom-up lipidomics has lead us to the notion that now both bottom-up and top-down analysis could be run on the same instrumentation platform.
’ MATERIALS AND METHODS Chemicals and Lipid Standards. Synthetic lipid standards and a total lipid extract of bovine heart were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) or Sigma-Aldrich Chemie (Munich, Germany); common chemicals and solvents of ACS or LCMS grade from Sigma-Aldrich Chemie (Munich, Germany) or Fluka (Buchs SG, Switzerland); methanol (LiChrosolv grade) from Merck (Darmstadt, Germany). Annotation of Lipid Species. Lipid classes: PE, phosphatidylethanolamines; PE-O, 1-O-alkyl-2-acylglycerophosphoethanolamines; LPE; lyso-phosphatidylethanolamines; PS, phosphatidylserines; PC, phosphatidylcholines; PC-O, 1-O-alkyl2-acylglycerophosphocholines; LPC, lysophosphatidylcholines; SM, sphingomyelins; PI, phosphatidylinositols; TAG, triacylglycerols; 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 fatty alcohol moietyæ:Æno. of double bonds in the first fatty acid or fatty alcohol moietyæ/Æno. of carbon atoms in the second fatty acid moietyæ:Æno. of double bonds in the second fatty acid moietyæ. For example, PC 18:0/18:1 stands for a phosphatidylcholine comprising the moieties of stearic (18:0) and oleic (18:1) fatty acids. If the exact composition of fatty acid or fatty alcohol moieties was unknown, the species were annotated as Ælipid classæ Æno. of carbon atoms in both moietiesæ:Æno. of double bonds in both moietiesæ. In this way, PC 36:1 stands for the PC species having 36 carbon atoms and one double bond in both 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 tissue grinder (Sigma-Aldrich Chemie, Munich) and extracted with 700 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 the MTBE/MeOH mixture prior to lipid extraction in the concentration of 10 μM each and the 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
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mixture was shaken for another 15 min. Phase separation was completed upon centrifuging for another 5 min in a benchtop centrifuge MiniSpin (Eppendorf, Hamburg) at 13 400 rpm. The upper organic phase was collected and stored at 25 C under nitrogen. Samples for Mass Spectrometric Analysis. Retina total lipid extracts or lipid standards were diluted with a mixture of isopropanol/methanol/chloroform 4:2:1 (v/v/v) containing 7.5 mM ammonium formate or ammonium acetate, as indicated separately for each experiment. Also, where specified, we used a mixture of methanol/chloroform 5:1 (v/v) containing 0.1% (v/v) triethylamine. Prior to the analysis, samples were loaded into a 96-well plate (Eppendorf, Hamburg), sealed with aluminum foil, and centrifuged for 5 min at 4 000 rpm on a Multifuge 3S-R centrifuge from Heraeus DJB Labcare Ltd. (Newport Pagnell, U.K.). The retina extract was diluted 10 times prior to the analysis. Final concentrations of synthetic standards were selected individually for each experiment. Mass spectrometric analses were performed on the LTQ Orbitrap XL (further termed as XL) and LTQ Orbitrap Velos (further termed as Velos) instruments (Thermo Fisher Scientific, Bremen), both equipped with a robotic nanoflow ion source TriVersa (Advion BioSciences, Ithaca NY) using chips with spraying nozzles with a diameter of 4.1 μm. The ion source was controlled by Chipsoft 6.4 software. The ionization voltage and gas backpressure were set to 1.25 kV and 0.95 psi in the positive and 0.7 kV and 1.06 psi in negative ion modes, respectively. Under these settings, 10 μL of 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 were 90 V (MSþ) or 150 V (MS) for both machines; and the s-lens level was 58% for the Velos. Isolation of Lipid Precursor Ions on XL and Velos Machines. The abundance of isolated precursors was monitored by the method of total ion mapping. To this end, MS/MS spectra were acquired with the ion trap (IT) or (where specified) with the Orbitrap (FT) analyzer, while the target precursor 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), and normalized collision energy (nCE) (1%) were fixed, while the target 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 a mixture of synthetic lipid standards LPC 12:0, PC 12:0/12:0, PC 14:0/14:0, 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 of 2.5 μM. Total ion maps were acquired under the following settings: nCE was 1%; isolation width was 1.5 Th; ITmax was 250 ms; target value for AGC was 5 000; and target mass resolution at 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 HCD modes was compared by acquiring collision energy profiles for the synthetic lipid standard PE 18:1/18:2. The analyte with a concentration of 0.5 μM was infused into a mass spectrometer in the negative ion mode, and the abundances of precursor and fragment ions were plotted against the normalized collision energy. MS/MS spectra were acquired under the following common settings: isolation width was 1.5 Th; ITmax was 100 ms; AGC 5481
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Analytical Chemistry value was set to 25 000; target mass resolution Rm/z400 was 30 000; for each spectrum, 3 scans were averaged within the total acquisition time of 1.19 s. Identification and Quantification of Molecular Species in Total Lipid Extracts. Centroided HCD FT MS/MS spectra were acquired in data-dependent mode as described.19 Each data-dependent acquisition (DDA) cycle consisted of one FT MS survey spectrum acquired at the target resolution Rm/z400 of 100 000, followed by the acquisition of five HCD FT MS/MS spectra at the resolution Rm/z400 of 30 000. The prescan option was disabled. Precursor ions were subjected to MS/MS if their m/z matched the masses in the inclusion list with the accuracy of (5 ppm. In MS/MS experiments, precursor ions were isolated at the linear ion trap with the isolation width of 1.6 Th, and the ITmax was set to 4 s. Exclusion time was set to 20 min. On average, a DDA experiment was completed in 40 min such that each precursor was fragmented twice. In HCD mode, nCE was 45%; fragments (m/z g 100) were detected by the Orbitrap analyzer at the resolution Rm/z400 of 30 000. The maximum scan time for acquiring one HCD FT MS/MS spectrum was 5.4 s. The lock mass option was enabled and abundant background anion of octadecyl-(di-tert-butyl-hydroxyphenyl)propionate ([M H]) with m/z 529.46262 was used 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 FT MS, ITmax was 100 ms and 3 scans were averaged for each spectrum. DDA experiments were repeated four times for each sample and twice for the blank. Lipid species were identified by LipidXplorer software as described in the Supporting Information, part 1. Lipid species were quantified by comparing the abundances of precursor peaks in high-resolution MS spectra and acyl anions peaks in MS/MS spectra with corresponding peaks of precursors and fragments of spiked internal standards.19,20 For quantifying rat retina lipids, abundances of monoisotopic peaks were adjusted according to isotopic profiles calculated from elemental compositions of corresponding molecular ions to compensate the large difference in the length of fatty acid moieties. LipidXplorer installer, MFQL libraries, and a lipid identification 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 Linear Ion Trap. If lipid species of the same class differ by a double bond
in their hydrocarbon moieties, their precursors are spaced by a m/z difference of 2 Th. Since in MS/MS experiments these precursors may produce the same specific fragment (like, the same headgroup), it would be advantageous to fragment their peaks separately, and therefore in data-dependent shotgun experiments precursors are typically isolated at the unit or higher mass resolution.19 To determine the abundance profile of precursor ions isolated at the unit resolution of the linear trap, we acquired MS/MS spectra of PC 16:0/22:6 in positive and negative ion modes with the XL and Velos. Normalized collision energy was maintained at 1% such that precursors remained intact. Note that in these experiments we could not employ internal standards to compensate for spraying instability, which typically contributed to less than 12% variation of the lipid peaks abundance (Supporting Information part 2, Figure S13).
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Figure 1. Relative abundance of the precursor ion of PC 16:0/22:6 isolated by the linear ion trap at the XL and Velos in (A) positive mode ([M þ H]þ; calculated m/z 806.5694) and (B) negative mode ([M þ HCOO]; calculated m/z 850.5593). In positive mode, the average precursor 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 average precursor m/z measured in MS experiments were 850.32 (0.24 Th) on IT; 806.5603 (0.10 ppm) on Orbitrap (FT) at the XL; and 850.5609 (0.62 ppm) on FT at the Velos. At the x-axis, the mass offset was calculated as the difference between the target and calculated precursor m/z and was not adjusted for the mass calibration; y-axis, the precursor ion abundance was normalized to the maximum value observed in each experiment. IT/IT stands for the MS/MS experiment in which the precursor ion was isolated and detected at the ion trap; IT/FT, precursor was isolated at the ion trap and detected at the Orbitrap.
To maximize the abundance of isolated precursors, a mass offset (the difference between the precursor m/z and m/z targeted in MS/MS experiment) should be applied on both the XL and Velos machines (Figure 1 and Supporting Information, part 2, Table S1). The unequal offset applied at XL and Velos machines could be explained by the difference in design of linear traps and ion isolation procedures.34 Further experiments with lipid standards suggested that at both XL and Velos, the mass offset did not change if AGC was set 5482
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Figure 2. Optimal offset value is independent of the lipid class and/or m/z of lipid species. Total ion maps were acquired from precursor ions of 11 synthetic lipid standards (names boxed) at nCE of 1% and isolation width of 1.5 Th. Optimal offset values correspond to the maximal abundance of isolated monoisotopic peaks. Measurements were performed in triplicate in negative ion mode on an XL machine.
to the higher target value (30 000 instead of 5 000 used to produce data for Figure 1). At the same instrument, the offset did not change between positive and negative modes and showed day-to-day variation within the range of 0.1 Th, which corresponded to one m/z step at the total ion map plot (Figure 1). For the same polarity, the offset was also unaffected by changing the isolation width within 1.0 to 1.8 Th, although the abundance of isolated 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 might degrade unstable precursors.36,37 Therefore, we further acquired total ion maps from precursors of 11 synthetic lipid standards and determined optimal offset values and their variations (Figure 2). With the isolation width of 1.5 Th, the optimal offset was, on average, 0.4 Th independently of the lipid class or m/z of the species and was within the error margin set by varying ion current and spraying stability. We therefore concluded that applying mass offsets is important for the unit isolation of lipid precursors. The actual offset values depend on the ion trap design but are weakly affected by acquisition polarity, AGC target values, width of the isolation window, as well as m/z and chemical structures of isolated lipid precursor. Once determined in experiments with reference substances, the same offset could be applied for profiling of multiple lipid classes. How isolation width affects the absolute abundance of precursor ions? Lipids are usually detected as molecular cations or anions but also as adducts with chloride or acetate anions in negative or lithium or ammonium cations in positive ion mode, respectively (reviewed in refs 8 and 10), which differ by their collisional stability. Therefore we fixed the mass offset and nCE and determined how the abundance of monoisotopic peaks was affected 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 the negative ion mode (Figure 3).
Figure 3. Abundance of isolated monoisotopic peaks depends on precursor ion stability. (A) Isolation of [M þ AcO] precursor ion of PC 16:0/22:6 detected by FT MS/MS at m/z 864.5740 (þ1.73 ppm); target m/z 865.0. (B) Isolation of [M H] precursor ion of PE 18:0/22:6 detected by FT MS/MS at m/z 790.5392 (þ1.39 ppm); target m/z 790.8. Experiments were performed on an LTQ Orbitrap XL instrument with a nCE 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 were calculated on the basis of three independent experiments while no reference signals compensating for the ion current instability were employed.
The monoisotopic peak of the molecular anion of PE could be isolated with no major abundance losses down to the width of 1.0 Th. In contrast, under the same settings the monoisotopic peak of the acetate adduct of PC was undetectable, while its intensity gradually increased with increasing the isolation width. Full MS/MS spectra contained no [M 15] fragment ions of demethylated PC.31 We reasoned that unit resolution isolation rapidly degraded the [M þ AcO] precursor, while its major collisional dissociation product [M 15] was not trapped. Although we could detect the peak of acetate adduct at the Velos even at the 1.0 Th isolation window, its abundance was 2.5-fold lower compared to the isolation at the “fully opened” window of 2.0 Th. We observed that molecular ions of several lipid classes, such as sphingolipids, suffered even larger abundance losses. It was 5483
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Figure 4. Normalized collision energy profiles of PE 18:0/18:1 (m/z 744.5549) produced by CID (panel A), PQD (panel B), and HCD (panel C) acquired on the Velos machine. m/z of fragment ions in the inset: m/z 283.2643, acyl anion of 18:0 fatty acid; m/z 281.2486, acyl anion of 18:1 fatty acid. NLs stands for the combined intensity of m/z 480.3091 and m/z 478.2933, the products of neutral loss of ketens of 18:1 and 18:0 fatty acids, respectively. TIC stands for the total ion current.
almost impossible to isolate their [M þ H]þ precursors within the width of less than 3 Th and their collisional instability
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(“fragility”, according to refs 36 and 37) remains a major bottleneck of bottom-up shotgun lipidomics experiments on ion trap instruments. In our experience, it could be alleviated by targeting different molecular ion forms, applying intrasource precursor ion separation38 and isolating ions at the broader width. HCD as a Resource for Lipid Species Profiling. Once precursor ions have been isolated in the ion trap, both the XL and Velos offer multiple ways for their subsequent fragmentation. They can be fragmented in the linear ion trap in the CID or PQD modes or in the HCD mode in the separate multipole collision cell attached to the C-trap. Here we examined the analytical merits of different fragmentation means for identifying molecular species of common glycerophospholipid classes. Molecular species of glycerophospholipids are usually identified by their accurate precursor masses and acyl anion fragments of their fatty acid moieties. Using the standard of PE 18:0/18:1, we compared the efficiency of precursor ion fragmentation and the 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 abundant acyl anion fragments were produced by HCD. First, HCD is a multiple collision process. Similar to the fragmentation in classical triple quadrupole instruments, higher CE drives the degradation of both precursors and intermediates down to the most stable acyl anion fragments. Second, the HCD cell C-trapOrbitrap scheme is more permissive toward transmission and detection of low-molecular weight fragments, compared to conventional ion traps:39 it is only limited by rf-amplitude of the C-trap and cuts off m/z below (approximately) 1/20 of the precursor m/z and therefore does not compromise the detection of acyl anion fragments. Quantitative profiling of glycerophospholipid species via acyl anions of their fatty acid moieties is an established analytical approach used on hybrid quadrupole time-of-flight mass spectrometers in multiple precursor ion scanning (MPIS)11,40 and datadependent acquisition (DDA)19 modes. Quantitative profiles obtained in independent DDA and MPIS experiments corroborate,19 and the linear dynamic range of species detection exceeds 10 000-fold.11,40 Here we examined if HCD of glycerophospholipid anions could be applied for molecular species profiling in the same way. We observed that in HCD mode, normalizing the collision energy offset enabled equally efficient fragmentation of the same class species, irrespective of their m/z (Supporting Information, part 2, Figure S3). Then we tested how the yield of acyl anions depended 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 compared to PC and PE, while maximum intensity for the acyl anions of all species was achieved at nCE of 65%. We then estimated the dynamic range of HCD quantification of lipids of different classes. Test samples were obtained by serial dilution of a mixture of seven synthetic lipid standards. Precursor masses were subjected to HCD FT MS/MS, and the combined intensity of peaks of acyl anions plotted against the species concentrations. We observed the linear response within better than a 1000-fold concentration range for each of the quantified standards (Supporting Information, part 2, Figure S5). 5484
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Analytical Chemistry We therefore concluded that, on instruments of the LTQ Orbitrap family, HCD outperformed CID and PQD in quantitative molecular species profiling. Normalization of the collision energy enabled one to achieve an equally complete yield of acyl anions from glycerophospholipid precursors independent of their 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
Figure 5. The abundance of acyl anion fragments of 16:0, 18:0, and 18:2 fatty 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 bovine heart extract on the XL instrument. The abundance of each fragment ion was normalized to its maximal value and represents the average of two independent experiments. While similar profiles were observed for other acyl anion fragments from different species, only 16:0, 18:0, and 18:2 are displayed here for presentation clarity. Collision energy profiles of major fragment ions of PC 34:2 and PE 36:2 are presented in the Supporting Information, part 2, Figure S4.
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and part per million mass accuracy enabled unequivocal assignment of fragment ions in MS/MS spectra and was particularly important for the quantification of lipids with polyunsaturated fatty acids (PUFA) moieties. In MS/MS, acyl anion fragments of PUFA species undergo further fragmentation with neutral loss of CO2.41,42 In this way, acyl anion of docosahexaenoic (22:6) fatty acid produces a fragment with calculated m/z 283.2431, which is isobaric to the acyl anion of abundant stearic (18:0) fatty acid (m/z 283.2643) (see the Supporting Information, part 2, Figure S6 for chemical structures). While their peaks overlap in lowresolution MS/MS spectra, they were completely resolved in FT MS/MS spectra (Figure 6) and therefore accurate quantification of the PUFA species did not rely upon arbitrary corrections of peak abundances,40 yet required careful selection of nCE (see below). Glycerophospholipidome of Rat Retina by HCD FT MS/MS. We applied HCD to profile a total lipid extract of rat retina, which is enriched in glycerophospholipid species with PUFA moieties.43 The analysis of, in total, 7 mg of tissue was performed in two biological replicas, each of which was analyzed in four technical replicas. Each shotgun analysis consumed 820 pmol of the total lipid material and produced a data set of 125 FT MS and 600 HCD FT MS/MS spectra acquired using the inclusion list of precursor m/z computed from expected elemental compositions of glycerophospholipid species.19,20 Because of the isolation width of 1.5 Th, isobaric precursors were fragmented together, while individual species were recognized and quantified in “mixed” MS/MS spectra by lipid class-specific and lipids species-specific fragment ions. Molecular species (Figure 7) were identified by LipidXplorer software (see the Supporting Information, part 1 for details). Bulk lipid class composition independently determined by HCD FT MS/MS and by FT MS in positive and negative modes (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 the abundance of individual molecular species, including species with very long chain (VLC) PUFA moieties.45 We underscore that, because of its focus on glycerophospholipidome, this
Figure 6. HCD FT MS/MS of PE 18:0/22:6 ([M H]) acquired at Rm/z400 of 30 000 on the XL machine. Peaks of the acyl anion of FA 18:0 and the product of CO2 loss from the acyl anion of FA 22:6 were fully resolved (inset). Chemical structures of fragment ions are presented in the Supporting Information, part 2, Figure S6. [FA 18:0] and [FA 22:6] designate acyl anion fragments of corresponding fatty acid moieties. 5485
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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 FT MS/MS did not distinguish ether lipids from plasmalogens.
analysis did not encompass other important lipid classes, such as sphingolipids or acylglycerols. Interestingly, we detected VLC PUFA species with 2634 carbon atoms and up to 6 double bonds in which both fatty acid moieties were highly unsaturated. As a representative example, HCD FT MS/MS spectrum of PC 32:6/22:6 is presented in Supporting Information, part 2, Figure S9. Despite that they are uncommon and relatively low abundant, their HCD identification was confident since it relied upon multiple fragments matched with sub-ppm accuracy. Since exact quantities of individual species in rat retina were unknown, we seek indirect means for validating that our quantitative assignments were correct. Lipid quantification method relying upon HCD FT MS/MS is conceptually similar to shotgun methodology established on a quadrupole time-of-flight mass spectrometer.19,20,40 While adapting it to another instrument, we checked if the combined abundances of acyl anions and their neutral loss products in MS/MS spectra independently corroborated the abundances of intact precursors in MS spectra for each lipid class. Indeed, we found that for the molecular species of four major classes (PC, PE, PI, and PS) average correlation coefficient r2 between the quantitative estimates based on FT MS and HCD FT MS/MS spectra was of the value of 0.992 (Supporting Information, part 2, Figure S10). High-resolution HCD FT MS/MS distinguished isobaric fragments and enabled direct quantification of species with 22:6 fatty acid moieties. To test if neutral loss products were identified correctly, we temporarily disabled accounting for CO2 loss fragments (see Supporting Information, part 1) and checked if the abundances of major PC species were affected. As expected, upon correction for neutral losses the content of PC species without PUFA was unchanged, 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 was required. At nCE > 50% we observed substantial uncompensated losses in the abundances of PUFA fragments, in contrast to acyl anions having zero to two double bonds (see the Supporting Information, part 2, Figure S12).Therefore, for accurate accounting of the PUFA-containing species nCE might be lower than the optimal value (Figure 5), despite overall detection sensitivity is slightly reduced. High resolution and mass accuracy of HCD FT MS/MS spectra were also instrumental in dissecting molecular composition of other lipid classes (in particular, triacylglycerols) by discriminating ether species from isobaric species comprising odd numbered fatty acid moieties (see the Supporting Information, part 2, Figure S13). Taken together, we demonstrated that HCD FT MS/MS supported accurate quantitative profiling of molecular species of a variety of lipid classes, including species comprising polyunsaturated or odd numbered fatty acid moieties.
’ CONCLUSIONS AND PERSPECTIVES In this work we established HCD, a fragmentation method recently introduced on hybrid LTQ Orbitrap tandem mass spectrometers, as a powerful tool for quantitative profiling of molecular species of glycerophospholipids. High mass resolution and relative abundance of structurally specific acyl anion fragments were the two important factors that increased both the confidence in molecular species assignment and quantification accuracy. We further demonstrated that LipidXplorer software took full advantage of the high mass resolution and mass accuracy in both MS and MS/MS spectra and supported their accurate and consistent interpretation. Therefore it could now be possible to perform both top-down and bottom-up lipidomics on a single instrumentation platform and support both approaches by the same software. 5486
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Analytical Chemistry This work also revealed important technical limitations of LTQ Orbitrap instruments. Despite marked improvements in the ion trap technology, isolation of precursors with unit or higher mass resolution was impacting unstable precursors. Although currently no generic solution is available, in many (yet, not all) instances it might be possible to minimize it by using larger width of isolation windows or analyzing alternative molecular forms (such as adducts) with enhanced collisional stability. In summary, the high mass resolution of tandem mass spectrometers in both MS and MS/MS modes, together with a palette of fragmentation methods and software, now brings the potential to understand the significance of the compositional complexity of lipidomes for cell biology and molecular medicine.14
’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT We are grateful for our colleagues at MPI CBG and the Technical University of Dresden for collaboration and support. We are particularly thankful to Dr. Christer S. Ejsing (University of Southern Denmark, Odense) for his valuable input and useful discussions and to Kathleen Eeisenhofer for critical reading of the manuscript. 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 was supported by the TRR 83 Grant from Deutsche Forschungsgemeinschaft (DFG) and the Virtual Liver (Code/0315757) Grant from Bundesministerium f. Bildung u. Forschung (BMBF). D.S. is supported by the Wellcome Trust/DBT India Alliance. ’ REFERENCES (1) Dennis, E. A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 2089–2090. (2) Oresic, M.; Hanninen, V. A.; Vidal-Puig, A. Trends Biotechnol. 2008, 26, 647–652. (3) Wenk, M. R. Cell 2010, 143, 888–895. (4) Gross, R. W.; Han, X. Chem. Biol. 2011, 18, 284–291. (5) van Meer, G. EMBO J 2005, 24, 3159–3165. (6) van Meer, G.; Voelker, D. R.; Feigenson, G. W. Nat. Rev. Mol. Cell. Biol. 2008, 9, 112–124. (7) Yetukuri, L.; Ekroos, K.; Vidal-Puig, A.; Oresic, M. Mol. Biosyst. 2008, 4, 121–127. (8) Griffiths, W. J.; Wang, Y. Chem. Soc. Rev. 2009, 38, 1882–1896. (9) Glish, G. L.; Burinsky, D. J. J. Am. Soc. Mass Spectrom. 2008, 19, 161–172. (10) Blanksby, S. J.; Mitchell, T. W. Annu. Rev. Anal. Chem. (Palo Alto Calif) 2010, 3, 433–465. (11) Ejsing, C. S.; Sampaio, J. L.; Surendranath, V.; Duchoslav, E.; Ekroos, K.; Klemm, R. W.; Simons, K.; Shevchenko, A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 2136–2141. (12) Kalvodova, L.; Sampaio, J. L.; Cordo, S.; Ejsing, C. S.; Shevchenko, A.; Simons, K. J. Virol. 2009, 83, 7996–8003. (13) Sampaio, J. L.; Gerl, M. J.; Klose, C.; Ejsing, C. S.; Beug, H.; Simons, K.; Shevchenko, A. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 1903–1907.
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