Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry
2014; Elsevier BV; Volume: 55; Issue: 8 Linguagem: Inglês
10.1194/jlr.m046995
ISSN1539-7262
AutoresAlan T. Maccarone, Jackson Duldig, Todd W. Mitchell, Stephen J. Blanksby, Eva Duchoslav, J. Larry Campbell,
Tópico(s)Analytical Chemistry and Chromatography
ResumoGlycerophospholipids (GPs) that differ in the relative position of the two fatty acyl chains on the glycerol backbone (i.e., sn-positional isomers) can have distinct physicochemical properties. The unambiguous assignment of acyl chain position to an individual GP represents a significant analytical challenge. Here we describe a workflow where phosphatidylcholines (PCs) are subjected to ESI for characterization by a combination of differential mobility spectrometry and MS (DMS-MS). When infused as a mixture, ions formed from silver adduction of each phospholipid isomer {e.g., [PC (16:0/18:1) + Ag]+ and [PC (18:1/16:0) + Ag]+} are transmitted through the DMS device at discrete compensation voltages. Varying their relative amounts allows facile and unambiguous assignment of the sn-positions of the fatty acyl chains for each isomer. Integration of the well-resolved ion populations provides a rapid method (< 3 min) for relative quantification of these lipid isomers. The DMS-MS results show excellent agreement with established, but time-consuming, enzymatic approaches and also provide superior accuracy to methods that rely on MS alone. The advantages of this DMS-MS method in identification and quantification of GP isomer populations is demonstrated by direct analysis of complex biological extracts without any prior fractionation. Glycerophospholipids (GPs) that differ in the relative position of the two fatty acyl chains on the glycerol backbone (i.e., sn-positional isomers) can have distinct physicochemical properties. The unambiguous assignment of acyl chain position to an individual GP represents a significant analytical challenge. Here we describe a workflow where phosphatidylcholines (PCs) are subjected to ESI for characterization by a combination of differential mobility spectrometry and MS (DMS-MS). When infused as a mixture, ions formed from silver adduction of each phospholipid isomer {e.g., [PC (16:0/18:1) + Ag]+ and [PC (18:1/16:0) + Ag]+} are transmitted through the DMS device at discrete compensation voltages. Varying their relative amounts allows facile and unambiguous assignment of the sn-positions of the fatty acyl chains for each isomer. Integration of the well-resolved ion populations provides a rapid method (< 3 min) for relative quantification of these lipid isomers. The DMS-MS results show excellent agreement with established, but time-consuming, enzymatic approaches and also provide superior accuracy to methods that rely on MS alone. The advantages of this DMS-MS method in identification and quantification of GP isomer populations is demonstrated by direct analysis of complex biological extracts without any prior fractionation. Differences in molecular structure are well understood to profoundly influence the biological function of glycerophospholipids (GPs). Numerous accounts have examined the role of GPs in cellular biochemistries including membrane permeability, protein aggregation, and receptor activation (1.GurrM. I.HarwoodJ. L.FraynK. N.. 2002. Lipid Biochemistry. Blackwell Science, Oxford. 215–263.Google Scholar, 2Lee A. Membrane structure.Curr. Biol. 2001; 11: R811-R814Abstract Full Text Full Text PDF PubMed Google Scholar, 3Janmey P.A. Kinnunen P.K.J. Biophysical properties of lipids and dynamic membranes.Trends Cell Biol. 2006; 16: 538-546Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 4Gross R.W. Jenkins C.M. Yang J.Y. Mancuso D.J. Han X.L. Functional lipidomics: the roles of specialized lipids and lipid-protein interactions in modulating neuronal function.Prostaglandins Other Lipid Mediat. 2005; 77: 52-64Crossref PubMed Scopus (41) Google Scholar, 5Menon A.K. Lipid modifications of proteins.in: Vance D. E Vance J. E In Biochemistry of Lipids, Lipoproteins and Membranes. 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Recent reports point to specific arrangements of acyl chains in GPs being responsible for structural interactions that induce specific activity. This has been noted particularly in the interactions of GPs toward nuclear receptor proteins. For example, Liu et al. (14Liu S. Brown J.D. Stanya K.J. Homan E. Leidl M. Inouye K. Bhargava P. Gangl M.R. Dai L. Hatano B. et al.A diurnal serum lipid integrates hepatic lipogenesis and peripheral fatty acid use.Nature. 2013; 502: 550-554Crossref PubMed Scopus (158) Google Scholar) examined the diurnal variation in fat metabolism in mice and suggested that the phosphatidylcholine (PC) (18:0/18:1), and not its isomer PC (18:1/18:0) (where the nomenclature indicates sn-1/sn-2 positions), acts as a trigger for the mediation of FA breakdown in muscles via PPARα signaling. Elsewhere, Ingraham and colleagues (15Krylova I.N. Sablin E.P. Moore J. Xu R.X. Waitt G.M. MacKay J.A. Juzumiene D. Bynum J.M. Madauss K. Montana V. et al.Structural analyses reveal phosphatidyl inositols as ligands for the NR5 orphan receptors SF-1 and LRH-1.Cell. 2005; 120: 343-355Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar) have reported the crystal structure of the phosphatidylglycerol (PG) (18:1/16:1) bound to the receptor steroidogenic factor 1 indicating that GP ligands with this arrangement of acyl chains on the glycerol backbone may be required for the protein to function in steroid synthesis. These, and related studies, have used CID to examine the acyl chain composition and glycerol backbone position of the target GPs. Such assignments rely on general trends in the product ion abundances in CID mass spectra and are based on literature precedent (16Hsu F-F. Turk J. Electrospray ionization with low-energy collisionally activated dissociation tandem mass spectrometry of glycerophospholipids: mechanisms of fragmentation and structural characterization.J. Chromatogr. B Analyt. Technol. 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Separation of GP sn-positional isomers by conventional reversed-phase LC, however, is only possible where one of the acyl chains has a high degree of unsaturation (21Nakanishi H. Iida Y. Shimizu T. Taguchi R. Separation and quantification of sn-1 and sn-2 fatty acid positional isomers in phosphatidylcholine by RPLC-ESIMS/MS.J. Biochem. 2010; 147: 245-256Crossref PubMed Scopus (67) Google Scholar). In the absence of rapid and definitive methods for the determination of acyl chain position, assigning sn-position in GPs is often based on the convention of the more unsaturated acyl chain occupying the sn-2 position (see below). This raises concerns that some reported GP structures may be entirely incorrect or ignore the likelihood of both isomers being present in the sample. Indeed, it has recently been suggested that GP notation be modified to reflect whether the sn-position of the acyl chains has been explicitly determined (22Liebisch G. Vizcaíno J.A. Köfeler H. Trötzmüller M. Griffiths W.J. Schmitz G. Spener F. Wakelam M.J.O. Shorthand notation for lipid structures derived from mass spectrometry.J. Lipid Res. 2013; 54: 1523-1530Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar). Current knowledge of the most common acyl chain distribution patterns within GPs has been developed over the past 40 years and is based primarily on digestion within lipid extracts (or subfractions thereof) by enzymes that hydrolyze the ester moieties at select positions on the glycerol backbone (23Stern W. Pullman M.E. Acyl-CoA-sn-glycerol-3-phosphate acyltransferase and positional distribution of fatty acids in phospholipids of cultured cells.J. Biol. Chem. 1978; 253: 8047-8055Abstract Full Text PDF PubMed Google Scholar). These techniques work well for determining the distribution of different FAs at the sn-1 and sn-2 of all GPs in an extract and/or a targeted subclass. Numerous accounts detailing the study of eukaryotic lipids have led to the convention of assigning saturated and unsaturated acyl chains at the sn-1 and sn-2 positions, respectively (24Kiełbowicz G. Gladkowski W. Chojnacka A. Wawrzeńczyk C. A simple method for positional analysis of phosphatidylcholine.Food Chem. 2012; 135: 2542-2548Crossref PubMed Scopus (25) Google Scholar, 25Connor W.E. Lin D.S. Thomas G. Ey F. DeLoughery T. Zhu N. Abnormal phospholipid molecular species of erythrocytes in sickle cell anemia.J. Lipid Res. 1997; 38: 2516-2528Abstract Full Text PDF PubMed Google Scholar, 26Van Deenen L.L. Chemistry of phospholipids in relation to biological membranes.Pure Appl. Chem. 1971; 25: 25-56Crossref PubMed Scopus (43) Google Scholar, 27Lands W.E.M. Stories about acyl chains.Biochim. Biophys. Acta. 2000; 1483: 1-14Crossref PubMed Scopus (123) Google Scholar). However, it should be noted that exceptions to this generality have been documented, such as the "unusual" sn-distribution in PGs from the bacterial strain Mycoplasma gallisepticum reported by Rottem and Markowitz (28Rottem S. Markowitz O. Membrane lipids of Mycoplasma gallisepticum: a disaturated phosphatidylcholine and a phosphatidylglycerol with an unusual positional distribution of fatty acids.Biochemistry. 1979; 18: 2930-2935Crossref PubMed Scopus (36) Google Scholar) where unsaturated fatty acyl chains were found to be prevalent at the sn-1 position. Enzymatic hydrolysis of complex lipid mixtures generally falls short of allowing sn-position assignment for a specific combination of fatty acyl chains within a GP subclass. It follows that structural assignment at this level could be achieved if the target lipid could be purified, or at least the pool of lipids significantly simplified, prior to the enzyme assay. Yoshikawa and coworkers (29Shinzawa-Itoh K. Aoyama H. Muramoto K. Terada H. Kurauchi T. Tadehara Y. Yamasaki A. Sugimura T. Kurono S. Tsujimoto K. et al.Structures and physiological roles of 13 integral lipids of bovine heart cytochrome c oxidase.EMBO J. 2007; 26: 1713-1725Crossref PubMed Scopus (299) Google Scholar) succeeded in this by examining only the lipids selectively bound to bovine heart cytochrome C oxidase upon crystallization. From this simplified pool, the GP component was further purified and subjected to both MS and phospholipase A2 (PLA2)-catalyzed hydrolysis. This analysis allowed definitive assignment of the acyl chain positions within the PG (16:0/18:1) associated with the protein. This approach, while definitive in assigning molecular structure to GPs, cannot be universally applied and requires significant sample amounts. Such results serve to highlight the need for a technique that can rapidly and unambiguously assign sn-position in GPs on diminishingly small amounts of crude lipid extract. Gas phase separation of isomeric compounds has been demonstrated using ion mobility spectrometry (IMS) with mixtures of ionized molecules resolved based on several physicochemical properties of the ions, including m/z, size and shape, and dipole moment (30Kanu A.B. Dwivedi P. Tam M. Matz L. Hill Jr, H.H. Ion mobility-mass spectrometry.J. Mass Spectrom. 2008; 43: 1-22Crossref PubMed Scopus (885) Google Scholar, 31EicemanG. A.KarpasZ.. 2005. Ion Mobility Spectrometry. CRC Press, Boca Raton, FL.Google Scholar). In a recent critical evaluation of the current tools for lipidomics, some of us have suggested that differentiation of isomeric lipids could be achieved by combining IMS and MS workflows (12Brown S.H.J. Mitchell T.W. Oakley A.J. Pham H.T. Blanksby S.J. Time to face the fats: what can mass spectrometry reveal about the structure of lipids and their interactions with proteins?.J. Am. Soc. 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Separation and classification of lipids using differential ion mobility spectrometry.J. Am. Soc. Mass Spectrom. 2011; 22: 1146-1155Crossref PubMed Scopus (93) Google Scholar) used a DMS device coupled to an ion-trap mass spectrometer to resolve the ionized lipid diacylglycerols (DGs) (16:0/12:0/OH) and (16:0/OH/12:0), which differ only in the position of the acyl chains on the glycerol backbone. Motivated by this demonstration of rapid gas phase sn-positional isomer separation on a planar DMS-MS platform, the workflow described herein was developed for the separation and relative quantitation of sn-positional isomeric PCs from complex biological extracts on a commercially available system that couples planar DMS with triple quadrupole ion-trap MS. The shorthand notation for lipids suggested recently by Liebisch and coworkers (22Liebisch G. Vizcaíno J.A. Köfeler H. Trötzmüller M. Griffiths W.J. Schmitz G. Spener F. Wakelam M.J.O. Shorthand notation for lipid structures derived from mass spectrometry.J. Lipid Res. 2013; 54: 1523-1530Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar) that builds on prior recommendations (41Fahy E. Subramaniam S. Brown H.A. Glass C.K. Merrill A.H. Murphy R.C. Raetz C.R.H. Russell D.W. Seyama Y. Shaw W. et al.A comprehensive classification system for lipids.J. Lipid Res. 2005; 46: 839-861Abstract Full Text Full Text PDF PubMed Scopus (1135) Google Scholar, 42Fahy E. Subramaniam S. Murphy R.C. Nishijima M. Raetz C.R.H. Shimizu T. Spener F. van Meer G. Wakelam M.J.O. Dennis E.A. Update of the LIPID MAPS comprehensive classification system for lipids.J. Lipid Res. 2009; 50: S9-S14Abstract Full Text Full Text PDF PubMed Scopus (1065) Google Scholar) is used extensively throughout this manuscript when denoting lipid structure. For example, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine is represented as PC (16:0/18:1), where "PC" represents the PC subclass of the GP class, the "18:1" indicates the number of carbon atoms:number of double bonds, and its placement after the forward slash assigns the position of esterification specifically at sn-2 on the glycerol backbone. Analogously, the 16:0 positioning before the forward slash indicates a palmitoyl chain esterified at the sn-1 position. Where the assignment of the sn-position of the acyl chains is uncertain or a mixture of both possible isomers is present, we have adopted the PC (16:0_18:1) notation (22Liebisch G. Vizcaíno J.A. Köfeler H. Trötzmüller M. Griffiths W.J. Schmitz G. Spener F. Wakelam M.J.O. Shorthand notation for lipid structures derived from mass spectrometry.J. Lipid Res. 2013; 54: 1523-1530Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar). Liebisch and coworkers do not recommend the use of parentheses unless specifying double bond position and stereochemistry within a specific acyl chain. However, in this manuscript parentheses are used to encapsulate a given sn-1 and sn-2 acyl chain pair to aid distinction between frequently mentioned sn-positional isomers. The term "regioisomer" is used exclusively in this manuscript to refer to one in a given pair of GP sn-positional isomers. Silver-adducted lipids denoted as [M + Ag]+ in this work refer to those formed from the 107Ag isotope unless otherwise specified. Synthetic PCs (16:0/18:1), (18:1/16:0), (16:0/18:0), (18:0/16:0), (18:0/18:1), (18:1/18:0), and (16:0/18:2) were obtained from Avanti Polar Lipids Inc. (Alabaster, AL). Silver acetate, lithium acetate, glycerol, sodium chloride, Tris base, PLA2 from honeybee venom (Apis mellifera), and the PC fraction from chicken egg yolk were obtained from Sigma-Aldrich (St. Louis, MO). Analytical grade ammonium acetate, calcium chloride, dichloromethane, and LC/MS-grade methanol were purchased from Thermo Fisher Scientific (Scoresby, Victoria, Australia), while distilled deionized water (18 MΩ) was produced in-house using a Synergy UV purification system (Millipore, North Ryde, New South Wales, Australia). All chemicals listed previously were used without further purification. Bovine (Bos taurus L.) brain and kidneys were collected from the Wollondilly Abattoir (Picton, New South Wales, Australia) immediately following the death of the animals, and the lipids were extracted as previously described (43Nealon J.R. Blanksby S.J. Mitchell T.W. Else P.L. Systematic differences in membrane acyl composition associated with varying body mass in mammals occur in all phospholipid classes: an analysis of kidney and brain.J. Exp. Biol. 2008; 211: 3195-3204Crossref PubMed Scopus (17) Google Scholar). All solutions were prepared for ESI and varied slightly in concentration depending on the experiment. In the positive-mode experiments, cow brain and kidney extracts were infused at a concentration of 0.1 μM total lipids in methanol containing 50 μM silver acetate, while the egg yolk PC fraction and synthetic lipid solutions contained 0.05 μM (also in 50 μM silver acetate in methanol). For the assay following enzymatic hydrolysis, the lysophosphatidylcholine (LPC) mixtures contained 0.4 μM total LPC in methanol doped with 5 mM ammonium acetate. Synthetic lipid mixtures and all extracts were made up to 0.05 μM total PC in 45:45:10 dichloromethane-methanol-water containing 12 mM ammonium acetate for use in negative-mode experiments. The flow rate in all cases was 15–20 μl min−1. A differential mobility spectrometer system (SelexIONTM, AB SCIEX, Concord, Ontario, Canada) was mounted in the atmospheric pressure region between the sampling orifice of a QTRAP® 5500 system (AB SCIEX) and a TurboVTM ESI source (37Schneider B.B. Covey T.R. Coy S.L. Krylov E.V. Nazarov E.G. Planar differential mobility spectrometer as a pre-filter for atmospheric pressure ionization mass spectrometry.Int. J. Mass Spectrom. 2010; 298: 45-54Crossref PubMed Scopus (131) Google Scholar, 39Campbell J.L. Le Blanc J.C.Y. Schneider B.B. Probing electrospray ionization dynamics using differential mobility spectrometry: the curious case of 4-aminobenzoic acid.Anal. Chem. 2012; 84: 7857-7864Crossref PubMed Scopus (83) Google Scholar). All mass spectral data were acquired and analyzed using AnalystTM software version 1.5.2. The following parameters were set unless noted otherwise. The ESI probe was maintained at 5,500 V, with a source temperature of 150°C, nebulizing gas pressure of 20 psi, and auxiliary gas pressure of 5 psi. Nitrogen was used as the curtain gas (20 psi), resolving gas (0 to 35 psi), and CID target gas with inlet set to 3 (arbitrary units, pressure ∼3 mTorr) for the MS2 and MS3 experiments. A constant gas flow in the DMS cell is achieved by the vacuum pumping of the MS system, and the DMS temperature was maintained at 225°C. The fundamental mechanisms and general operation of this particular form of DMS have been described elsewhere (31EicemanG. A.KarpasZ.. 2005. Ion Mobility Spectrometry. CRC Press, Boca Raton, FL.Google Scholar, 37Schneider B.B. Covey T.R. Coy S.L. Krylov E.V. Nazarov E.G. Planar differential mobility spectrometer as a pre-filter for atmospheric pressure ionization mass spectrometry.Int. J. Mass Spectrom. 2010; 298: 45-54Crossref PubMed Scopus (131) Google Scholar, 39Campbell J.L. Le Blanc J.C.Y. Schneider B.B. Probing electrospray ionization dynamics using differential mobility spectrometry: the curious case of 4-aminobenzoic acid.Anal. Chem. 2012; 84: 7857-7864Crossref PubMed Scopus (83) Google Scholar, 44Shvartsburg A.A. Differential Ion Mobility Spectrometry: Nonlinear Ion Transport and Fundamentals of FAIMS. CRC Press, Boca Raton, FL2009: 1-293Google Scholar). Typically, the DMS was operated at a fixed optimal separation voltage (SV = 4,100 V), while the compensation voltage (CV) was ramped from +9 to +14 V. During each 0.10 V step in CV, data were acquired in either an MS2 (enhanced product ion) or MS3 mode. In either case, five scans were summed at each CV step for a total acquisition time of 4 to 5 min. The resulting ionograms were smoothed once using a Gaussian algorithm with a 1 point width prior to extracting peak areas. Manual integration was necessary in some cases where the signal intensity of a feature was relatively low. In complex extracts where the ions representing [PC (34:2) + 109Ag]+ and [PC (34:1) + 107Ag]+ were isobaric, isotope corrections were carried out as described in supplementary Fig. V. In certain experiments, both SV and CV were set for the collection of CID spectra from Q1-isolated precursor ions {m/z 866.5 for [PC (16:0_18:1) + Ag]+}, and in these cases, data were acquired for 3 min. In between different samples, the syringe and line were flushed with solvent to eliminate cross-contamination of results, which was verified by observing no analytical signal while sampling a blank solution. The QTRAP 5500 has been modified for ozone-induced dissociation (OzID) in a similar fashion as described previously (45Poad B.L.J. Pham H.T. Thomas M.C. Nealon J.R. Campbell J.L. Mitchell T.W. Blanksby S.J. Ozone-induced dissociation on a modified tandem linear ion-trap: observations of different reactivity for isomeric lipids.J. Am. Soc. Mass Spectrom. 2010; 21: 1989-1999Crossref PubMed Scopus (115) Google Scholar). Here, a combination CID/OzID workflow was used as this has been shown capable of revealing acyl chain sn-position in phospholipids (13Pham H.T. Maccarone A.T. Thomas M.C. Campbell J.L. Mitchell T.W. Blanksby S.J. Structural characterization of glycerophospholipids by combinations of ozone- and collision-induced dissociation mass spectrometry: the next step towards "top-down" lipidomics.Analyst. 2014; 139: 204-214Crossref PubMed Google Scholar). Sodiated PC (16:0_18:1) cations were generated by ESI and were selected in Q1 at m/z 782.2. These ions were accelerated into q2 [collision energy (CE) = 38 eVLab, i.e., the value set for CE in AnalystTM that corresponds to the voltage offset between the q0 and q2 rods] where they could fragment upon collisions with target gas consisting of a mixture of N2, O3, and O2. The fragment ions, as well as residual intact PC ions, were then trapped in q2 for 1 s. Product ions from the CID/OzID fragmentation processes were cooled and transferred to Q3, where they were analyzed by mass-selective axial ejection at 10,000 Th s−1. All spectra reported here represent the average over 50 scans, totaling 1 min of acquisition time. Experiments were also conducted with the QTRAP 5500 using the MS3 workflow developed by Ekroos et al. (18Ekroos K. Ejsing C.S. Bahr U. Karas M. Simons K. Shevchenko A. Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation.J. Lipid Res. 2003; 44: 2181-2192Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) to determine relative regioisomeric content of PCs. Ions of m/z 818.5 corresponding to [PC (16:0_18:1) + CH3COO]− were formed during negative ion ESI. The
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