title: 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 E. 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.
authors.affiliation: Department of Chemistry, Vassar College, Poughkeepsie, NY 12604, USA. tegarrett@vassar.edu
authors.forename: Teresa A
authors.initials: TA
authors.lastname: Garrett
authors.forename: Christian R H
authors.initials: CR
authors.lastname: Raetz
authors.forename: Jennifer D
authors.initials: JD
authors.lastname: Son
authors.forename: Travis D
authors.initials: TD
authors.lastname: Richardson
authors.forename: Craig
authors.initials: C
authors.lastname: Bartling
authors.forename: Ziqiang
authors.initials: Z
authors.lastname: Guan
mesh_terms: D000214:Acyl Coenzyme A / Q000032:analysis / Q000737:chemistry; D002853:Chromatography, Liquid; D045424:Complex Mixtures / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry / Q000302:isolation & purification*; D013058:Mass Spectrometry; D010705:Phosgene / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000032:analysis / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000214:Acyl Coenzyme A; D045424:Complex Mixtures; D008055:Lipids; D010714:Phosphatidylethanolamines; D010705:Phosgene; C483858:phosphatidylethanolamine; C012046:succinyl-coenzyme A