Non-enzymatically derived minor lipids found in Escherichia coli lipid extracts

Abstract

Electrospray ionization mass spectrometry is a powerful technique to analyze lipid extracts especially for the identification of new lipid metabolites. A hurdle to lipid identification is the presence of solvent contaminants that hinder the identification of low abundance species or covalently modify abundant lipid species. We have identified several non-enzymatically derived minor lipid species in lipid extracts of Escherichia coli, phosphatidylmethanol, ethyl and methyl carbamates of PE and N-succinyl PE were identified in lipid extracts of Escherichia coli. Phosphatidylmethanol (PM) was identified by exact mass measurement and collision induced dissociation tandem mass spectrometry (MS/MS). Extraction in the presence of deuterated methanol leads to a 3 atomic mass unit shift in the [M-H]- ions of PM indicating its formation during extraction. Ethyl and methyl carbamates of PE, also identified by exact mass measurement and MS/MS, are likely to be formed by phosgene, a breakdown product of chloroform. Addition of phosgene to extractions containing synthetic PE significantly increases the levels of PE-MC detected in the lipid extracts by ESI-MS. Extraction in the presence of methylene chloride significantly reduced the levels of these lipid species. N-succinyl PE is formed from reaction of succinyl-CoA with PE during extraction. Interestingly N-succinyl PE can be formed in an aqueous reaction mixture in the absence of added E. coli proteins. This work highlights the reactivity of the amine of PE and emphasizes that careful extraction controls are required to ensure that new minor lipid species identified using mass spectrometry are indeed endogenous lipid metabolites.

Colistin Treatment Affects Lipid Composition of Acinetobacter baumannii

“…Other glycerophospholipids detected in previous studies [ 32 , 35 ] such as phosphatidic acid (PA), phosphatidylserine (PS), lysophosphatidylglycerol (LPG), hydroxylated-PE, CL, and MLCL (PE-OH, CL-OH, and MLCL-OH) were not detected in our conditions. However, phosphatidylmethanol (PMeOH) lipids were annotated but, since these minor lipids are formed during lipid extraction, they were not considered [ 41 ]. Interestingly, PC was annotated with six different species.…”

Identification of a novel cationic glycolipid in Streptococcus agalactiae that contributes to brain entry and meningitis

“…The acyl chain compositions and exact masses of Lys-Glc-DAG molecular species observed in COH1 are listed in Table 2. “*” denotes methylcarbamate of Lys-Glc-DAG, an extraction artifact due to the use of chloroform (57).…”

“…S1. "*" denotes methylcarbamate of Lys-Glc-DAG , an extraction artifact due to the use of chloroform (60). and incubated at 28°C with rotation at 225 rpm for h. Cultures were pelleted using a Sorvall RC6+ centrifuge at 4,280 x g for 6 min at room temperature.…”

Ethanol Induced Brain Lipid Changes in Mice Assessed by Mass Spectrometry

“…However, PM and other phosphatidylalcohols are not native lipid species. Rather, they are generated during the extraction process from glycerophospholipids. Indeed if not deactivated by heat, in the presence of alcohols, the phosphatidyltransferase activity of phospholipase D causes artifactual formation of non-native lipids such as PM. , Even if the PM detected in this study are not directly biologically relevant (methanol was used during the extraction process), they give us interesting information about Phospholipase D activity.…”

“…However, PM and other phosphatidylalcohols are not native lipid species. Rather, they are generated during the extraction process 51 from glycerophospholipids. Indeed if not deactivated by heat, in the presence of alcohols, the phosphatidyltransferase activity of phospholipase D causes artifactual formation of non-native lipids such as PM.…”