Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation

Abstract

The molecular composition of phosphatidylcholines (PCs) in total lipid extracts was characterized by a combination of multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer and MS3 fragmentation on an ion trap mass spectrometer. Precursor ion spectra for 50 acyl anion fragments of fatty acids (fatty acid scanning) acquired in parallel increased the specificity and the dynamic range of the detection of PCs and identified the fatty acid moieties in individual PC species. Subsequent analysis of detected PC peaks by MS3 fragmentation on an ion trap mass spectrometer quantified the relative amount of their positional isomers, thus providing the most detailed and comprehensive characterization of the molecular composition of the pool of PCs at the low-picomole level. The method is vastly simplified, compared with conventional approaches, and does not require preliminary separation of lipid classes or of individual molecular species, enzymatic digestion, or chemical derivatization. The approach was validated by the comparative analysis of the molecular composition of PCs from human red blood cells.In the total lipid extract of Madin-Darby canine kidney II cells, we detected 46 PC species with unique fatty acid composition and demonstrated that the presence of positional isomers almost doubled the total number of individual molecular species. The molecular composition of phosphatidylcholines (PCs) in total lipid extracts was characterized by a combination of multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer and MS3 fragmentation on an ion trap mass spectrometer. Precursor ion spectra for 50 acyl anion fragments of fatty acids (fatty acid scanning) acquired in parallel increased the specificity and the dynamic range of the detection of PCs and identified the fatty acid moieties in individual PC species. Subsequent analysis of detected PC peaks by MS3 fragmentation on an ion trap mass spectrometer quantified the relative amount of their positional isomers, thus providing the most detailed and comprehensive characterization of the molecular composition of the pool of PCs at the low-picomole level. The method is vastly simplified, compared with conventional approaches, and does not require preliminary separation of lipid classes or of individual molecular species, enzymatic digestion, or chemical derivatization. The approach was validated by the comparative analysis of the molecular composition of PCs from human red blood cells. In the total lipid extract of Madin-Darby canine kidney II cells, we detected 46 PC species with unique fatty acid composition and demonstrated that the presence of positional isomers almost doubled the total number of individual molecular species. Phosphatidylcholines (PCs) are the most abundant glycerophospholipids in mammalian cells, accounting for more than 30% of the total lipid content (1Kawai K. Fujita M. Nakao M. Lipid components of two different regions of an intestinal epithelial cell membrane of mouse.Biochim. Biophys. Acta. 1974; 369: 222-233Crossref PubMed Scopus (135) Google Scholar). PCs comprise a glycerol phosphate backbone with a choline head group attached at the sn-3 position and two fatty acid moieties at positions sn-1 and sn-2. PCs are rather heterogeneous and have diverse physical properties, because they engage a variety of fatty acids having different numbers of carbon atoms and double bonds. In mammalian cells, PCs are synthesized via the CDP-choline pathway or via the phosphatidylethanolamine methylation pathway (2Cui Z. Vance J.E. Chen M.H. Voelker D.R. Vance D.E. Cloning and expression of a novel phosphatidylethanolamine N-methyltransferase. A specific biochemical and cytological marker for a unique membrane fraction in rat liver.J. Biol. Chem. 1993; 268: 16655-16663Abstract Full Text PDF PubMed Google Scholar, 3Kent C. Eukaryotic phospholipid biosynthesis.Annu. Rev. Biochem. 1995; 64: 315-343Crossref PubMed Scopus (313) Google Scholar, 4Walkey C.J. Yu L. Agellon L.B. Vance D.E. Biochemical and evolutionary significance of phospholipid methylation.J. Biol. Chem. 1998; 273: 27043-27046Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Further metabolism involves remodeling processes, which are controlled by the coordinated action of acyltransferases, transacylases, and lipases (5Yamashita A. Sugiura T. Waku K. Acyltransferases and transacylases involved in fatty acid remodeling of phospholipids and metabolism of bioactive lipids in mammalian cells.J. Biochem. (Tokyo). 1997; 122: 1-16Crossref PubMed Scopus (239) Google Scholar). Inhibiting the CDP-choline pathway is lethal (6Cui Z. Houweling M. Chen M.H. Record M. Chap H. Vance D.E. Terce F. A genetic defect in phosphatidylcholine biosynthesis triggers apoptosis in Chinese hamster ovary cells.J. Biol. Chem. 1996; 271: 14668-14671Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 7Baburina I. Jackowski S. Apoptosis triggered by 1-O-octadecyl-2-O-methyl-rac-glycero-3- phosphocholine is prevented by increased expression of CTP:phosphocholine cytidylyltransferase.J. Biol. Chem. 1998; 273: 2169-2173Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) and is not compensated by the alternative phosphatidylethanolamine methylation pathway, presumably because different PC species are produced (8DeLong C.J. Shen Y.J. 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Certain cellular compartments and membrane microdomains are distinguished by a characteristic composition of PCs, in which very long chain, or saturated, or highly unsaturated fatty acid moieties prevail (15Igbavboa U. Hamilton J. Kim H.Y. Sun G.Y. Wood W.G. A new role for apolipoprotein E: modulating transport of polyunsaturated phospholipid molecular species in synaptic plasma membranes.J. Neurochem. 2002; 80: 255-261Crossref PubMed Scopus (58) Google Scholar, 16Ramanadham S. Hsu F. Zhang S. Bohrer A. Ma Z. Turk J. Electrospray ionization mass spectrometric analyses of phospholipids from INS-1 insulinoma cells: comparison to pancreatic islets and effects of fatty acid supplementation on phospholipid composition and insulin secretion.Biochim. Biophys. Acta. 2000; 1484: 251-266Crossref PubMed Scopus (37) Google Scholar, 17Simons K. Toomre D. Lipid rafts and signal transduction.Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5187) Google Scholar, 18Schuck S. Honsho M. 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USA. 1997; PubMed Scopus Google Scholar). of the of the choline head PCs are detected in ion with the B. G. analysis of membrane lipids at the by ionization mass Natl. Acad. Sci. USA. 1997; PubMed Scopus Google Scholar). of of PCs a characteristic of the having which their in lipid extracts by precursor ion scanning B. G. analysis of membrane lipids at the by ionization mass Natl. Acad. Sci. USA. 1997; PubMed Scopus Google Scholar). the fragmentation of the PCs and molecular of PCs have of have not detected in be very and does not the fatty acid composition of PCs the total number of carbon atoms and double in fatty acid be from the of PC is not to which fatty acids in PCs and to the relative amount of their molecular quadrupole time-of-flight mass precursor ion scanning spectra for a number of I. A. B. to quadrupole time-of-flight mass PubMed Scopus Google Scholar). The of on mass is by the C. P. J. and of from by and ion and 2002; PubMed Scopus Google Scholar), was by in the cell and into the I. for a quadrupole time-of-flight mass spectrometer and its for precursor ion Biochem. 2000; Scopus Google Scholar). with the of quadrupole I. A. B. to quadrupole time-of-flight mass PubMed Scopus Google Scholar, K. Simons K. Shevchenko A. of phospholipids by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass Chem. 2002; PubMed Scopus Google Scholar), which are the most in mass spectrometric analysis of lipids for characterization of in Scholar). of of glycerophospholipids abundant acyl of fatty acid moieties Electrospray ionization mass analysis of human plasma membrane Natl. Acad. Sci. USA. PubMed Scopus Google Scholar, of molecular species of glycerophospholipids and mass Lipid Full Text PDF PubMed Google Scholar, Turk J. fragmentation in acids A 2000; PubMed Scopus Google Scholar, Turk J. and fragmentation in a 2000; PubMed Scopus Google Scholar, Turk J. of and by ionization mass a 2000; PubMed Scopus Google Scholar, Turk J. on with quadrupole mass with fragmentation and Scopus Google Scholar). of acyl anion fragments for multiple precursor ion scanning on a the of the fatty acid composition of individual glycerophospholipids in lipid a method fatty acid scanning K. Simons K. Shevchenko A. of phospholipids by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass Chem. 2002; PubMed Scopus Google Scholar). glycerophospholipids that are in ion with and is not to the fatty acid composition of a and the position of fatty acid moieties or in be from the of of peaks of acyl the relative a precursor is a of species, which might to the or to different classes of In we an for of PCs by a combination of on a mass spectrometer and MS3 fragmentation on an ion trap mass spectrometer. of in the detection of PCs which be by MS3 fragmentation of on an ion trap mass spectrometer to the relative of positional The analysis of lipid extracts from Madin-Darby canine kidney II cells and human red blood cells characterized the pool of PCs a of and species. PC from and from and from Cell from was from and was from from from II cells in and in at in a on to II cells with and in a for at at Cell in and at red blood cells by for at at and with and into and at Lipid was to and M. A method for the and of total lipids from Biol. Chem. Full Text PDF PubMed Google Scholar). was to a cell and for by of and for The was for at at The was to a new and was by the for at for at The was to a new and with The and the was to a new The was in a and at PC and their by by M. of van in and Molecular Scholar). of PC was in and with of and for and in a and for mass spectrometric analysis The concentration of PCs in was by the by and G. S. A. separation of lipids and of phospholipids by analysis of 1970; 5: PubMed Scopus Google Scholar). in different and in or total lipid extracts from II cells and from red blood cells in to mass spectrometric in an at a concentration of Precursor ion scanning was on a mass spectrometer with a ion K. Simons K. 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In and ion the quadrupole was at and with a of of of the in the was to the of the and was controlled via The characteristic ion of was for detection of PCs and in ion of PC precursor ion spectra of the two acyl anion fragments of the fatty acid moieties analysis of lipid precursor ion spectra acquired for acyl anion fragments of fatty acid to carbon atoms and to double bonds. was at in and ion an of to of the spectra a of fragmentation was on a quadrupole ion trap mass spectrometer with a ion of the was into and at to The of the was the was and the was of and MS3 precursor an of and The relative was between and positional isomers of PCs, we the of the sn-2 in PCs, specificity by of and the by detected the signal from atoms of the not that from the which that the sn-1 was not to not that the the of the sn-2 by is spectrometric analysis of PC on a mass spectrometer by and by that they a amount of positional isomers in in by and ion trap MS3 fragmentation of the PCs is in and of lipid fragmentation of in ion of of the lipid of the of for was and for ion trap and fatty acid quadrupole of the lipid of the of for was and for ion trap MS3 fragmentation of in ion in a new fatty acid quadrupole a detection of PCs in ion is or into an of PCs anion of of phospholipids via ionization mass 1995; PubMed Scopus Google Scholar, of molecular species of glycerophospholipids and mass Lipid Full Text PDF PubMed Google Scholar, A. S. T. of phospholipids mass PubMed Scopus Google Scholar) anion of PC a group from the choline and the a ion with than the mass of the of the PC The fragmentation via of abundant by of fatty acid or of or by acyl of fatty acids A of was in analysis of PCs of of phospholipids via ionization mass 1995; PubMed Scopus Google Scholar) of glycerophospholipids on a quadrupole Turk J. fragmentation in acids A 2000; PubMed Scopus Google Scholar, Turk J. and fragmentation in a 2000; PubMed Scopus Google Scholar, Turk J. of and by ionization mass a 2000; PubMed Scopus Google Scholar, Turk J. on with quadrupole mass with fragmentation and Scopus Google Scholar). The acyl anion of sn-2 fatty acid was more abundant than the fatty acid at sn-1 the in which the of fragments on the spectra by the precursor ion and the of the the of precursor and with of acyl anion The of acyl at and presumably because and of the ion in the mass spectrometer. In the range of the almost on the of acyl of the was for the acyl of fatty acid moieties are produced by of anion of PCs, they be by to classes of glycerophospholipids K. Simons K. Shevchenko A. of phospholipids by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass Chem. 2002; PubMed Scopus Google Scholar). analysis of and was by and to acyl of acid and acid and precursor of and detected in the in spectra of the of the precursor ion was more abundant in for the acyl anion of sn-2 fatty acid than of sn-1 fatty The of their not on the of analysis and the in the of positional The of of precursor peaks in different precursor was in the concentration range and was not PC into a lipid extract not PC to which demonstrated that and not Further analysis of precursor ion demonstrated that produced a of the of positional isomers of the fragmentation of anion of PCs on the mass spectrometer not that the relative of PC species In with mass with a cell quadrupole or ion trap mass a precursor ion by an which ion be and in of the mass spectrometric in to quadrupole ion trap mass 1997; Scopus Google anion of PCs in an ion trap mass spectrometer of positional fragmentation of the of produced abundant of a of PC at MS3 fragmentation of in of the fatty acids of at and with the of the the most anion fragments at and although they abundant than in fragments by of fatty acid from detected at and fragmentation of the produced the acyl anion and of and fatty acid at and not trap analysis of PC the fragmentation not MS3 fragmentation of the produced a in which the of compared with the of that fragmentation in ion trap and mass are that of fatty acids acyl or is a MS3 fragmentation of PC abundant the of sn-2 fatty acid that be in of and species. the relative of with the of of species by of The of of via of fatty acids with the of by the of detection the we of in various of the by and the content of positional isomers on the mass spectrometer. of the of by MS3 and the demonstrated a between the The was of and and produced not the that of fatty acid at the sn-2 The of fragments or a combination of of fragments in MS3 spectra not with the of the isomers, most because the of fragments from fragmentation is than that of Further we that acid fragments by of from the sn-2 position of in of and although they abundant than produced by of fatty acid K. and A. 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T. phospholipid molecular species of in cell Lipid 1997; Full Text PDF PubMed Google Scholar). identified molecular species of PCs by and species identified in the by a combination of and that we not precursor ion spectra for acyl of a fatty acids although the by and by molecular species species of and their relative content was PC species with relative content that by detected by mass with relative ion trap MS3 analysis of PC species that positional isomers by are in human red blood cells having a highly unsaturated acid at the sn-1 of the total molecular composition of PCs from human red blood number of carbon number of double number of carbon number of double in a new detected PC species a fatty acid with an number of carbon that and are fatty acids with an number of that are in PCs from red blood cells, and we detected in molecular species that and of the molecular composition of PCs by and by the mass The II cell is an for the of epithelial and the of and lipid we a combination of and MS3 fragmentation to the molecular composition of PCs in a total lipid Lipid extracts of II cells in ion by to The total number of carbon atoms and double in fatty acid moieties was from of detected The was by in ion precursor ion spectra of acyl of fatty acids to the of by which to the fatty acid composition of detected PC species molecular composition of PCs from II trap number of carbon number of double in fatty acid number of carbon number of double in fatty acid in a new by to precursor ion a at was detected by was not the to because with the of abundant at identified species and In total lipid extract of II cells, we identified PC species positional fatty acid moieties with an number of carbon although was rather the of de novo lipid biosynthesis in mammalian cells. ion trap MS3 analysis on the most abundant PCs that of and MS3 analysis the fatty acid and to that and and of the total with species accounting for to that the presence of a of positional isomers in PCs is to a rather than a specific of a II cell that of PC species with fatty acids having an number of carbon atoms might with of PCs, in which an is to the glycerol backbone via an rather than an in the of ion at was not of lipids not be by and not the of in PC peaks in which a acyl anion was and in which the fatty acid with an number of carbon atoms was not the presence of species. and ion trap MS3 fragmentation of the molecular composition of PCs by a variety of and species. 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T. of phospholipids mass PubMed Scopus Google Scholar) The different in and ion trap and in by which the is to precursor and the of is not that the fragmentation of PC in a at acyl anion fragments of fatty abundant peaks of in MS3 ion trap The of fragmentation by ion trap MS3 that they have different positional specificity of fatty acid for of the total of and almost via the of sn-2 fatty acid of thus does not on the fatty acid is from the of of species different of fatty acids and anion fragments are abundant in MS3 spectra of the total ion and are not produced from the sn-2 fatty acid of of acyl anion of fatty acid to be a that for of the total ion of and PC species by ion trap MS3 fragmentation is and does not require The is very specific because of the of precursor at and MS3 and is to total lipid of and acyl anion fragments of sn-2 fatty acid the the MS3 fragmentation is for the detection of PC in lipid for the of PC species with different molecular precursor ion scanning is on ion trap mass analysis by mass and by ion trap mass be in on a mass spectrometer and MS3 fragmentation on the ion trap mass spectrometer to the molecular composition of the method of and not increased the specificity and the dynamic range of detection to the fatty acid moieties and their relative on the glycerol backbone of the individual PC MS3 fragmentation of precursor detected by of the relative of their positional a combination of mass spectrometric for individual molecular species of PCs, we that is the most detailed and comprehensive approach for their A combination of and MS3 was to PCs in a lipid extract from II cells. The conventional analysis by detected peaks of PCs Further analysis of the by that they 46 species with unique fatty acid MS3 fragmentation of the most abundant peaks that they are of positional isomers, and the total number of molecular species is to that although we detected amount of PCs, we not that they might be in the extract C. D. of choline is for the of Madin-Darby canine kidney cells.J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) by more abundant species. PCs of II cells and human red blood cells have of the presence of positional isomers of phospholipid metabolism in rat for of J. Biochem. PubMed Scopus Google Scholar). Biochemical have that fatty acid remodeling is a in (5Yamashita A. Sugiura T. Waku K. Acyltransferases and transacylases involved in fatty acid remodeling of phospholipids and metabolism of bioactive lipids in mammalian cells.J. Biochem. (Tokyo). 1997; 122: 1-16Crossref PubMed Scopus (239) Google Scholar), which for the presence of lipid species. have demonstrated that positional isomers different of and have in A. phospholipids with with in Chem. B. 2000; Scopus Google Scholar, A.D. A. in effect of acyl relative and Rev. 1998; Scopus Google Scholar). positional isomers in their to with thus the of membrane the the role of the be in molecular the molecular composition of PCs of by mass conventional A combination of head group scanning and ion trap MS3 fragmentation the most detailed and comprehensive characterization of the molecular the relative of individual molecular species. The of detection was at the level. The was vastly and not require preliminary separation of lipid classes and of individual molecular species, enzymatic digestion, or chemical derivatization. In of acyl be with thus the dynamic range and the specificity of detection of lipid glycerophospholipids fatty acid moieties of very long fatty acids S. Hsu F. Zhang S. Bohrer A. Ma Z. Turk J. Electrospray ionization mass spectrometric analyses of phospholipids from INS-1 insulinoma cells: comparison to pancreatic islets and effects of fatty acid supplementation on phospholipid composition and insulin secretion.Biochim. Biophys. Acta. 2000; 1484: 251-266Crossref PubMed Scopus (37) Google Scholar), fatty acids having an number of carbon atoms C. D. T. U. fatty acids in J. 2000; PubMed Scopus Google Scholar), be detected and quantified in total extracts in the presence of of lipids with fatty acid is that the for the approach in which molecular species of multiple lipid classes be identified and quantified by of a very number of acquired precursor ion for of characteristic not the fragments of head and acyl of a variety of fatty The are to the of and of Molecular Cell and for and The are to and for a in of quadrupole time-of-flight mass The C. and for of the and of for enzymatic by fatty acid scanning mass of a PC phosphatidylcholine precursor ion scanning scanning for precursor that a ion with quadrupole time-of-flight acyl anion of a fatty acid with PC with fatty acid at sn-1 position of the glycerol and fatty acid at sn-2 position PC a of fatty acids and at position of the backbone