Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.

Abstract

Electrospray ionization mass spectrometry (ESI-MS) was utilized for the structural determination and qmntitative analysis of individual phospholipid molecula spedes from subplcomole amounts of human erythrocyte plasma membrane phospholipids. The sensitivity of ESI-MS was 2-3 orders of magnitude greater than that achievable with fastatom bombardment mass sectrometry (FAB-MS). Phospho- (10)(11)(12).It is now apparent that cellular activation is mediated, in large part, through the generation of lipid second messengers resulting from the activation of a multiplicity of different phospholipases (13-16). Accordingly, identification of the temporal course and extent of hydrolysis of specific phospholipid molecular species during cellular activation is a prominent question in signal transduction research. However, difficulties inherent in the structure determination and quantitation of subpicomole amounts of zwitterionic phospholipids present in biologic samples have thus far precluded facile analyses. Prior studies have demonstrated that electrospray ionization mass spectrometry (ESI-MS) allows access to a vast array of heretofore inaccessible problems in protein chemistry through dramatically enhanced volatilization of molecular ions (17)(18)(19)(20). However, to date, the utility of ESI-MS for the structure identification and quantitative analysis of phospholipids present in biologic membranes has not been realized. We now report that ESI facilitates the structural determination and quantitative analyses of individual molecular species of synthetic and naturally derived phospholipids with a sensitivity 2 to 3 orders of magnitude greater than that previously achieved by FAB-MS. Since this ionization modality is not complicated by differential fragmentation and idiosyncratic surface desorption, it will allow the direct quantitation and structural analysis of phospholipids from extracts of biologic samples. MATERIALS AND METHODSMaterials. Commercially available phospholipids were purchased from Avanti Polar Lipids or Nu Chek Prep (Elysian, MN). 1-O-(Z)-Hexadec-l'-enyl-2-octadec-9'-enoyl-snglycero-3-phosphocholine and 1-O-hexadecyl-2-octadec-9'-enoyl-sn-glycero-3-phosphocholine were prepared as described (21). All phospholipids and fatty acids were quantitated by capillary gas chromatography after acid methanolysis (22).ESI-MS. ESI mass spectra were acquired on a triplequadrupole tandem mass spectrometer (Finnigan MAT TSQ 700, San Jose, CA) equipped with an electrospray interface (Analytica of Branford, Branford, CT). The first and third quadrupoles of the instrument serve as independent analyzers and the second is a nonlinear octapole, which is utilized as a collision cell for MS-MS experiments. The detector of the instrument is an off-axis continuous dynode electron multiplier operable from -400 to -3000 V, with a variable postacceleration/conversion dynode voltage from -3 kV to -20 kV or +3 kV to +20 kV for detection of positive or negative ions, respectively. The effective mass range was from m/z 10 to m/z ...

Total structure characterization of unsaturated acidic phospholipids provided by vicinal di-hydroxylation of fatty acid double bonds and negative electrospray ionization mass spectrometry

“…In accordance with previous reports, all the PL species investigated in the present study produced abundant [M Ϫ H] Ϫ ions upon ESI [1][2][3][4][5][6][7][8][9][10]. Fragmentation reactions involving heteroatoms are favored because of the lower critical energy of such reactions and this applies especially to the low-energy regime of tandem quadrupole MS. CAD analysis of deprotonated PLs represents no exception to this principle.…”

“…This is recognized in the CAD production spectra of 16:0/11,12-(OH) 2 (Figures 4b and 6e, respectively). This is probably due to the preferred formation of the sn-1 carboxylate for PA [1][2][3][4] and PS [1,3,4,8,10], whereas sn-2 carboxylates are preferred for PG [1,3,[5][6][7]. The CAD product-ion spectrum of the 6,7-(OH) 2 -18:0 carboxylate is different from the other derivatized mono-unsaturated FAs as the product-ions resulting from Pathways 1a and 1b in Scheme 1 are both of low abundance.…”

“…Given that R x is the sn-1 (R 1 ) and the sn-2 (R 2 ) FA of the PL, respectively, the following series of five different product-ions were produced upon CAD of the [1][2][3][4][5][6][7][8][9][10]. Deprotonated PA, PG, PI, and PS species all show a preferred loss of the sn-2 FA either as a neutral FA or as a neutral ketene (product-ions 1-4) upon CAD [1][2][3][4][5][6][7][8][9][10], which are useful for assigning FA regioisomerism.…”

“…The structural diversity of PLs is attributable to different polar head groups, FA substituents, and their regioisomerism. These characteristics can be identified by investigating the deprotonated molecules of the PLs by negative electrospray ionization (ESI) tandem quadrupole mass spectrometry (MS) as demonstrated for phosphatidic acid (PA) [1][2][3][4], phosphatidylglycerol (PG) [1,3,[5][6][7], phosphatidylinositol (PI) [1, 3, 6, 8, 9,], and phosphatidylserine (PS) [1,3,4,8,10] species. However, a challenging task is to determine the double bond position(s) of unsaturated fatty acids, which are not identified by ESI-MS/MS of the deprotonated native species due to very limited COC cleavage(s) along the alkenyl chain.…”

Desorption electrospray ionization (DESI) mass spectrometry and tandem mass spectrometry (MS/MS) of phospholipids and sphingolipids: Ionization, adduct formation, and fragmentation

“…The mass spectrometric analysis of phosphatidylcholines (1 Scheme 1) has been studied extensively using FAB [9,36] and ESI [11,[37][38][39][40][41]. With a quaternary amine in its head group, GPCho ionizes well in the positive ion mode and generally poorly in the negative ion mode.…”