Constant neutral loss scanning for the characterization of bacterial phospholipids desorbed by fast atom bombardment

Abstract

Fast atom bombardment (FAB) of the lysed cells and crudellpld extracts of bacterla results In the selective desorption of phorphoupkk that are characteristic of bacterlal specks. I n thk paper, constant neutral loss (CNL) llnked scarmlng of the magnetk and electrlc sector flekls of a double focuslng mass spectrometer Is evaluated as an approach to the analysls of bacterla cells that can provlde greater selectlvlty for polar llplds and speclflclty for partlcular phosphollpld classes. I n the metastable t h e frame, neutral losses of phosphorylethanolamlne, phosphorylglycerol, and other polar head groups are be8t obtalned from protonated molecular Ions analyzed In the posltlve Ion mode. SelectlvHy Is Improved (relathre to normal magnetk fleld scans) for phosphollplds of a partlcular class In the presence of more abundant phosphollpMs and the background Ion dgnal characteristk of FAB m a s spectra. I n addklon, CNL scans penntl the observation of momnnethy@hoaphattklylethanolamhre, a mhror pbsphoUpkl prewnt In a few bacterla, such as P. vu&8f/s, but lndlstlngulshabk In mass from fatty acld homologues of PE contalnIng addltlonal methylene groups. Slnce fatty acld content In bacterial lipids (and therefore the masses appearlng In a particular spectrum) reflects growth condltlons as well as specks, the ldenttflcatlon of specllc phosphollpld classes by CNL scans provlcks a promlskrg approach to the klentllicatlon and cladlcatlon of bacterla.Membrane lipids are important biomarkers that can be used to differentiate bacteria from one another (1-3). We have found that desorption mass spectrometry techniques selectively ionize polar membrane lipids directly from lysed bacteria to provide mass spectra that are characteristic of Gram stain and species (4, 5). Both phospholipids and glycolipids are selectively desorbed by these techniques. The most common phospholipids contain two fatty acids, a glycerolphosphate core, and a polar head group which determines the phospholipid class (Scheme I). The variable portions of phospholipids (the head group and the fatty acids) are both found in distribution patterns that are chemotaxonomically significant.Thus, one approach to characterizing bacteria involves the chromatographic analysis of fatty acids extracted from bacteria cultured and harvested under standard conditions (6). Fatty acids are identified by retention times and their distribution is determined from the relative peak areas. While there are limits to the range of fatty acids that a given species will produce, the fatty acid distribution itself can vary significantly in the same species grown on different media, at different Scheme I -CO-O-CH, I CO-0-CH CH, I L/ 0-t-0 R J J R -CHiCH<NH, PE -CH,-CHOH-CH,OH PG -CH$HOH-CH,O-LYSINE LPG -INOSITOL PI -cn,-cn,-wicH, MMPE -cH,-CH,-N(cH,), DMPE temperatures or harvested in different growth phases (2).Hence the requirement for standard culture conditions. On the other hand, the presence and relative abundance of particular classes of phospholipids in a given bacteria are less sensit...

Flow-injection electrospray ionization mass spectrometry of crude cell extracts for high-throughput bacterial identification

“…The use of lipid based biomarkers for taxonomic purposes has to involve careful consideration of the growth conditions, as changes in the fatty acid composition to varying culture conditions would influence the spectral signal, as already observed with FAB-MS [39]. However, it is known from the FAB-MS literature (e.g., [39,45]) that neutral losses due to the loss of polar head groups from phospholipids and glycolipids can form a more stable basis for identification purposes. For example, in the present study Br.…”

Alcalilimnicola halodurans gen. nov., sp. nov., an alkaliphilic, moderately halophilic and extremely halotolerant bacterium, isolated from sediments of soda-depositing Lake Natron, East Africa Rift Valley.

“…In phospholipids such as PG or PC, cleavages that are characteristic of the polar head are known to occur in the negative and positive FAB modes (Heller et al, 1988). The resulting ions from such phospholipids have the structures of glycerophosphatic acids (GPA) and can be observed in the normal FAB as well as in the CID spectra (e.g.…”

Applications of MALDI‐TOF Mass Spectrometry in Clinical Diagnostic Microbiology

“…In the 1980s, the development of desorption/ionization techniques such as plasma desorption (PD), laser desorption (LD) and fast atom bombardment (FAB) allowed the generation of molecular biomarker ions from microorganisms leading to bacterial profiling (Heller et al, 1987;Platt et al, 1988). In early experiments, only biomarker molecules of low molecular masses such as bacterial lipids were analysed (Shah & Collins, 1980;Heller et al, 1988), as the processes used for the ionization of biomolecules were too energy rich to avoid unpredictable analyte decomposition. The evolution of soft ionization techniques such as MALDI and electrospray ionization (ESI) developed in the late 1980s (Tanaka & Fenn, 2002) made possible analysis by MS of large biomolecules such as intact proteins.…”