Comprehensive analysis of phospholipids and glycolipids in the opportunistic pathogen Enterococcus faecalis.

Enterococcus faecalis is a Gram-positive, opportunistic, pathogenic bacterium that causes a significant number of antibiotic-resistant infections in hospitalized patients. The development of antibiotic resistance in hospital-associated pathogens is a formidable public health threat. In E. faecalis and other Gram-positive pathogens, correlations exist between lipid composition and antibiotic resistance. Resistance to the last-resort antibiotic daptomycin is accompanied by a decrease in phosphatidylglycerol (PG) levels, whereas multiple peptide resistance factor (MprF) converts anionic PG into cationic lysyl-PG via a trans-esterification reaction, providing resistance to cationic antimicrobial peptides. Unlike previous studies that relied on thin layer chromatography and spectrophotometry, we have performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) directly on lipids extracted from E. faecalis, and quantified the phospholipids through multiple reaction monitoring (MRM). In the daptomycin-sensitive E. faecalis strain OG1RF, we have identified 17 PGs, 8 lysyl-PGs (LPGs), 23 cardiolipins (CL), 3 glycerophospho-diglucosyl-diacylglycerols (GPDGDAG), 5 diglucosyl-diacylglycerols (DGDAG), 3 diacylglycerols (DAGs), and 4 triacylglycerols (TAGs). We have quantified PG and shown that PG levels vary during growth of E. faecalis in vitro. We also show that two daptomycin-resistant (DapR) strains of E. faecalis have substantially lower levels of PG and LPG levels. Since LPG levels in these strains are lower, daptomycin resistance is likely due to the reduction in PG. This lipidome map is the first comprehensive analysis of membrane phospholipids and glycolipids in the important human pathogen E. faecalis, for which antimicrobial resistance and altered lipid homeostasis have been intimately linked.

Differentiation between peptides containing acetylated or tri-methylated lysines by mass spectrometry: an application for determining lysine 9 acetylation and methylation of histone H3.

Histone acetylation and methylation play a critical role in transcription and gene regulation. Identification of sites of lysine acetylation and methylation in histones or other proteins by mass spectrometry (MS) is of increasing interest. In this paper, we report the use of MS to differentiate between peptides containing acetylated or tri-methylated lysines. High accuracy matrix-assisted laser desorption/ionization-time of flight MS gives better than five parts per million measurement accuracy, which is sufficient to verify acetylation and/or methylation. Electrospray ionization tandem mass spectrometry was used to assign modification sites and to differentiate acetylation from methylation. Typically, an immonium ion at m/z 98 corresponds to a mono-methylated lysine and an immonium ion at m/z 126 corresponds to an acetylated lysine. The neutral loss ion (MH(+)-59) is unique for a tri-methylated lysine. For a peptide with two or more modification sites of acetylation or tri-methylation or one site containing partial acetylation and tri-methylation, the a(2)-, b(2)-type ion is the characteristic index for an acetylated lysine whereas the b(2)-59 ion is indicative of a tri-methylated lysine in the N-terminus. The y-type ions and y-59 ions are characteristic of an acetylated lysine and a tri-methylated lysine at the C-terminus, respectively. We demonstrated that a lysine in a peptide modified by methylation or acetylation can be differentiated by MS using our method. Even if more then one lysine is present in a peptide and different modifications of this amino acid occur, they can be distinguished. This method was successful for the determination of the acetylation and methylation status of lysine 9 of histone H3 in chicken erythrocytes and human HeLa cell lines.