Characterization and multiplexed quantification of derivatized aminophospholipids

Abstract The structural characterization and quantification of changes in cellular lipid abundances under different physiological or pathological states can provide critical information toward understanding the role of lipids in health and disease. Here, in order to develop a strategy for the simultaneous (i.e., multiplexed) relative quantification of aminophospholipids from within two different crude lipid extracts, we first evaluated the gas-phase fragmentation behavior of a series of sulfonium ion-containing S , S ′-dimethylthiobutanoylhydroxysuccinimide (DMBNHS) derivatized diacyl- and alkyl ether-linked aminophospholipids, and DMBNHS and iodine/methanol derivatized plasmalogen-aminophospholipids. Using positive ionization mode nESI coupled with a range of tandem mass spectrometry (MS/MS) and multistage-MS/MS (i.e., MS 3 ) collisional activation conditions, as well as high resolution/accurate mass measurements, the fragmentation pathways giving rise to characteristic product ions required for the collective, or individual, identification of each of these lipid sub-classes was determined. Then, based on the knowledge obtained by characterization of these fragmentation reactions, the ‘proof of principle’ application of this method was demonstrated by analysis of a 1:1 mixture of crude lipid extracts from a metastatic colorectal cancer cell line, SW620, and its alkylglycerone phosphate synthase (AGPS) siRNA knockdown, derivatized separately with isobaric mass stable isotope labeled d 6 -‘heavy’ and d 6 -‘light’ DMBNHS reagents. Relative quantification at the ‘sum composition’ level was shown to be readily achieved by high resolution/accurate mass MS/MS, by ratiometric measurement of pairs of abundant isotopically encoded ‘reporter’ ions formed via the neutral loss of S(CD 3 ) 2 and S(CH 3 ) 2 from the ‘heavy’ and ‘light’ derivatized aminophospholipids. Additional ‘molecular level’ structural characterization and secondary quantification of isomeric mixtures of O -alkenyl ether-containing plasmalogen-aminophospholipids were also achieved directly from the MS/MS spectra, via the observation and ratiometric measurement of additional pairs of isotopically encoded product ions. For further structural characterization and secondary quantification of diacyl- and alkyl ether-containing aminophospholipids, MS 3 dissociation of the initial neutral loss reporter ions was performed.