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Journal of Lipid Research, Vol. 45, 1355-1363, July 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Methods

Bacterial phospholipid molecular species analysis by ion-pair reversed-phase HPLC/ESI/MS

Nicolas Mazzella^*, Josiane Molinet^*, Agung Dhamar Syakti^*, Alain Dodi, Pierre Doumenq^1,*, Jacques Artaud^* and Jean-Claude Bertrand

^* Laboratoire de Chimie Analytique de l'Environnement, Unité Mixte de Recherche 6171, Institut Fédératif de Recherche Pôle Méditerannéen des Sciences de l'Environement 112, Europôle de l'Arbois, BP 80, 13545 Aix-en-Provence Cedex 4, France
Laboratoire d'Analyses Radiochimiques et Chimiques, Commissariat à l'Énergie Atomique, Centre d'études de Cadarache, 13108 St Paul lez Durance, France
Laboratoire de Microbiologie, Géochimie et Ecologie Marine, UMR 6117, case 901, Université de la Méditerranée, 163 avenue de Luminy, 13228 Marseille Cedex 09, France

¹ To whom correspondence should be addressed. e-mail: pierre.doumenq{at}univ.u-3mrs.fr

This work set out to optimize the detection and separation of several phospholipid molecular species on a reversed-phase column with the use of an electrospray ionization/mass spectrometry-compatible counter-ion. An application of this technique concerned a qualitative and quantitative analysis of bacterial membrane phospholipids extracted from Corynebacterium species strain 8. The phospholipid classes of strain 8 were identified as phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol, and a peculiar lipid compound, acyl phosphatidylglycerol.

Most of the molecular species structures were elucidated, and regarding phosphatidylglycerol, the fatty acid positions were clearly determined with the calculation of the sn-2/sn-1 intensity ratio of the fatty acyl chain fragments.

Abbreviations: APG, acyl phosphatidylglycerol; DMDS, dimethyl disulfide; DMPG, 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol; DPG, diphosphatidylglycerol; DPPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; ELSD, evaporative light-scattering detector; ESI, electrospray ionization; FAME, fatty acid methyl ester; IPMS, intact phospholipid molecular species; MSM, mineral salt medium; PC, phosphatidylcholine, PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PLFA, phospholipid ester-linked fatty acid; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol; PS, phosphatidylserine; TBAAc, tetrabutyl ammonium acetate; TBABr, tetrabutyl ammonium bromide; UV, ultraviolet light

Supplementary key words reversed-phase liquid chromatography/electrospray ionization/mass spectrometry • negative ionization • counter-ion • sn-1 and sn-2 fatty acid positions • Corynebacterium species

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