Utility of tBDMS-derivativatization and Software-Enhanced Mass Resolution for GC-MS Identification of Hydroxy Fatty Acids in Complex Bacterial Lipid Mixtures.

摘要

Hydroxy fatty acids (OH-FA) are significant components of bacterial lipopolysaccharides (LPS) and may affect LPS potency as a stimulator of the host immune system. Opportunistic and pathogenic bacteria, such as Pseudomonas aeruginosa (PA), adapt to environmental conditions, altering gene expression, production of secreted materials, and possibly LPS OH-FA composition. While fatty acid methyl esters (FAMEs) are readily identified by GC-MS, OH-FAMEs do not typically give strong spectral fingerprints, making them particularly difficult to identify in lipid mixtures. t-butyldimethylsilyl (tBDMS) derivatization of hydroxy fatty acids in bacterial FAME preparations, prior to GC-MS analysis markedly improved detection, and when combined with enhanced mass resolution and elemental analysis by post-acquisition software, tBDMS-derivatization permitted ready identification of hydroxy fatty acids in complex lipid mixtures. Bacterial fatty acid methyl esters (FAMEs) were either purchased, or derived from fresh cultures of P. aeruginosa (PA). PA FAMEs were obtained by direct treatment of whole cells with methanolic sulfuric acid, extracting into hexane, then hexane-ethyl ether. Following drying, no further fractionation was required. Bacterial FAMEs dissolved in chloroform-hexane were analyzed directly by GC-MS and, in parallel, subjected to derivatization with N-methyl-N-tert-dimethylsilyltrifluoroacetamide (MTBSTFA) and N-tert-butyldimethylsilyl-imidazole (TBSIM) prior to analysis. Chromatographic separation with mass spectral analysis was performed on a Varian CP-3800 GLC (VF-5MS capillary column, 30 M X 0.25 mm I.D. X 0.25 (mu)m film) interfaced with a Varian 1200L triple quadrupole mass spectrometer. Centroid and profile El spectra were obtained at -70eV in the positive ion mode. Calibrated masses and peak shapes were obtained from profile data with post-acquisition software MassWorks (Cerno Bioscience). Calibrated lineshape isotope profile searches (CLIPS) were performed for elemental analysis (MassWorks).