title: Rapid isolation and characterization of bacterial lipopeptides using liquid chromatography and mass spectrometry analysis.
abstract: The structural analysis of post-translational modifications (PTMs) of lipoproteins is difficult due to the hydrophobic properties of their fatty acid moieties. At the present time, the relative positions of fatty acid components on the N-acyl-S-diacylglycerylcysteine core structure has not been specifically identified in any natural or bacterial expressed recombinant lipoproteins. In this study, we describe a rapid solid-phase extraction using acetonitrile and isopropanol method that can be performed manually to isolate large amounts of relatively pure lipopeptides generated by the limited tryptic-digestion of recombinant lipoproteins. Using these lipopeptides and LC/MS mass spectra analysis, two groups of N-terminal lipidated (diacyl or triacyl) molecules that differ by one fatty acid unit were successfully identified. This LC/MS method also provided the separation of lipopeptides differing by 14 Da for the on-line MS identification. Multiple-stage fragmentation analyses of the di- and triacyl lipopeptides using both the positive and negative ion modes enabled to identify the putative structure of the N-acyl-S-diacylglycerylcysteine containing an amide bond to palmitic acid at the N-terminal cysteine, a palmitic acid at sn1 position, and an unsaturated fatty acid of either hexadecenoic acid, cyclopropaneoctanoic acid, oleic acid and nonadecenoic acid at sn2 position of diacylglycerol residue through ester bonding. For diacyl lipoprotein, the saturated palmitoyl fatty acid group is absent at sn1 position of glycerol-derived lipid residue of lipopeptide.
authors.affiliation: Vaccine Research and Development Center, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan, ROC.
authors.forename: Yan
authors.initials: Y
authors.lastname: Kwok
authors.forename: Wang-Chou
authors.initials: WC
authors.lastname: Sung
authors.forename: Annie Li-Hsiu
authors.initials: AL
authors.lastname: Lin
authors.forename: Hsueh-Hung
authors.initials: HH
authors.lastname: Liu
authors.forename: Frieda Ai-Hsiang
authors.initials: FA
authors.lastname: Chou
authors.forename: Steve Shih-Yang
authors.initials: SS
authors.lastname: Hsieh
authors.forename: Chih-Hsiang
authors.initials: CH
authors.lastname: Leng
authors.forename: Pele
authors.initials: P
authors.lastname: Chong
mesh_terms: D001419:Bacteria / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D055666:Lipopeptides / Q000737:chemistry* / Q000302:isolation & purification*; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D055666:Lipopeptides; D008074:Lipoproteins; D011994:Recombinant Proteins