Elucidation and identification of amino acid containing membrane lipids using liquid chromatography/high-resolution mass spectrometry

RATIONALE: Intact polar lipids (IPLs) are the building blocks of cell membranes, and amino acid containing IPLs havebeen observed to be involved in response to changing environmental conditions in various species of bacteri a. High-performance liquid chromatography/mass spectrometry (HPLC/MS) has become the primary method for analysis ofIPLs. Many glycerol-free amino acid containing membrane lipids (AA-IPLs), which are structurally different thanabundant aminophospholipids, have not been characterized using HPLC/MS. This results in many lipids remainingunrecognized in IPL analysis of microbial cultures and environmental samples, hampering the study of their occurrenceand functionality.METHODS: We analyzed the amino acid containing IPLs of a number of bacteria (i.e. Gluconobacter cerinus,Cyclobacterium marinus, Rhodobacter sphaeroides,andPedobacter heparinus) in order to decipher fragmentation pathways,and explore potential novel lipid structures using HPLC/electrospray ionizati on ion trap MS (HPLC/ESI-IT-MS) andHPLC/high-resolution MS (HPLC/HRMS).RESULTS: We report differentiation between glutamine and lysine lipids with the same nominal masses, novel MSfragmentation pathways of cytolipin, the lipopeptides cerilipin and ?avolipin, head group hydroxylated ornithine lipids,and the novel identi?cation of cerilipin with a hydroxylated fatty acid.CONCLUSIONS: Non-glycerol AA lipids can be readily recognized as their fragmentation follows a clear pattern withinitial dehydration or other loss from the head group, followed by fatty acid losses resulting in a diagnostic fragmention. Higher level MSnand HRMS are valuable tools in characterizing AA lipid head group structural components