Revealing Fatty Acid Heterogeneity in Staphylococcal Lipids with Isotope Labeling and RPLC–IM–MS

Abstract

Up to 80% of the fatty acids in Staphylococcus aureus membrane lipids are branched, rather than straight-chain, fatty acids. The branched fatty acids (BCFAs) may have either an even or odd number of carbons, and the branch position may be at the penultimate carbon (iso) or the antepenultimate (anteiso) carbon of the tail. This results in two sets of isomeric fatty acid species with the same number of carbons that cannot be resolved by mass spectrometry. The isomer/isobar challenge is further complicated when the mixture of BCFAs and straight-chain fatty acids (SCFAs) are esterified into diacylated lipids such as the phosphatidylglycerol (PG) species of the S. aureus membrane. No conventional chromatographic method has been able to resolve diacylated lipids containing mixtures of SCFAs, anteiso-odd, iso-odd, and iso-even BCFAs. A major hurdle to method development in this area is the lack of relevant analytical standards for lipids containing BCFA isomers. The diversity of the S. aureus lipidome and its naturally high levels of BCFAs present an opportunity to explore the potential of resolving diacylated lipids containing BCFAs and SFCAs. Using our knowledge of lipid and fatty acid biosynthesis in S. aureus, we have used a stable-isotope-labeling strategy to develop and validate a 30 min C18 reversed-phase liquid chromatography method combined with traveling-wave ion mobility–mass spectrometry to provide resolution of diacylated lipids based on the number of BCFAs that they contain.

Profiling of branched-chain fatty acids via nitroxide radical-directed dissociation integrated on an LC-MS/MS workflow

“…6 Hines and co-workers recently demonstrated the abundant presence of diacylated lipids containing BCFAs in Staphylococcus aureus . 7 For mammals, BCFAs are rich in lipids in skin, 8 secretions, 9 and vernix caseosa of newborns. 10 Aside from endogenous synthesis, the dietary intake of BCFAs, comprising approximately 2% of dairy fat, is the principal source of the BCFAs for the human body.…”

Do Nanodisc Assembly Conditions Affect Natural Lipid Uptake?

“…For each separation, approximately 5–8 μL of lipid samples was injected and trapped on a Waters XBridge C8 Direct Connect HP (10 μm, 2.1 × 30 mm) followed by separation on a Waters ACQUITY Premier CSH C18 UPLC column (1.7 μm, 1 × 100 mm). The mobile phase consisted of 60/40 acetonitrile/water with 10 mM ammonium acetate (mobile phase A, MPA) and 90/10 isopropanol/acetonitrile with 10 mM ammonium acetate (mobile phase B, MPB) . The 21 min gradient elution method was run at a flow rate of 0.25 mL/min and is described in Table S1.…”

“…The mobile phase consisted of 60/40 acetonitrile/ water with 10 mM ammonium acetate (mobile phase A, MPA) and 90/10 isopropanol/acetonitrile with 10 mM ammonium acetate (mobile phase B, MPB). 39 The 21 min gradient elution method was run at a flow rate of 0.25 mL/min and is described in Table S1.…”

Defective pgsA contributes to increased membrane fluidity and cell wall thickening in S. aureus with high-level daptomycin resistance

“…To investigate the source of elevated BCFAs in N315-D8, we evaluated the distribution of the branched short-chain acyl-CoA precursors to BCFAs using an LC-MS method (see Supplementary Methods in SI document). The choice of culture medium is known to influence the distribution of BCFA:SCFA in staphylococcal lipids, with growth in MHB yielding higher levels of BCFAS:SCFAs than S. aureus grown in TSB (28,32). As expected, N315 produced less significantly less C 5-CoA (representing both anteiso and iso form) when grown in TSB compared to MHB (SI Figure S6) while the amount of C4-CoA (branched and straight) was unchanged by the growth conditions.…”