Characterization of Bacterial Phospholipids by Electrospray Ionization Tandem Mass Spectrometry

Abstract

A negative ion electrospray ionization tandem mass spectrometric technique was developed for the analysis of glycerophospholipids. Examination of the product ion mass spectrum of the deprotonated molecular ion provided sufficient information to identify both the class of glycerophospholipid and the molecular weights of the two fatty acid moieties. This technique was applied to the profiling of glycerophospholipids present in the chloroform/methanol extracts of four different bacterial species. The principal bacterial phospholipids detected by this technique were phosphatidylglycerols and diphosphatidylglycerols, accompanied by small amounts of phosphatidylethanolamines for two of the bacterial species examined. The fatty acid composition of the phosphatidylglycerols for each bacteria was determined by tandem mass spectrometry and presented graphically. Differences in the fatty acid composition for each bacterial species were readily apparent from a visual examination of the data sets.

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.…”

“…Additionally, R 1 COO Ϫ and R 2 COO Ϫ are observed at m/z 255 (100%) and 281 (53%) respectively. These ions and their abundances are in accordance with previous reports [1][2][3][4]. A mechanism for the formation of R 2 COO Ϫ is shown in Scheme 1 (upper part) [2,4,7] which applies to all PL species constituting both native and derivatized FAs.…”

“…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.…”