MALDI-TOF-MS USING DIFFERENT MATRICES EXPOSES THE HETEROGENEITY OF LIPID A SPECIES PRESENT IN BACTERIAL LIPOPOLYSACCHARIDES

ADRIANA CASABUONO ; ALICIA S. COUTO

Los Cocos- Cordoba

Congreso; I Congreso Argentino de Espectrometría de masa; 2012

SAEM

Lipopolysaccharides (LPSs) are characteristic components of the outer membrane of the overwhelming majority of Gram-negative bacteria. LPS consists of lipid A, a core oligosaccharide that includes the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue, and an O-antigen polysaccharide also called the O-antigen composed of an oligosaccharide repeating unit. The O-antigen polymer glycosylates the core oligosaccharide which at its reducing end contains the Kdo residue linked to the lipid A. This latter is encrusted into the phospholipid layer. LPSs play a key role in the pathogenesis and in the toxic manifestation of Gram-negative infection promoting the activation of the immune system. In the present work we have performed for the first time a detailed profiling of the whole lipid A obtained from Shigella flexneri variant X LPS by matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). Different matrices were assayed in positive- and negative-ion modes (nor-harmane, CMBT, and also the system CMBT with EDTA ammonium salt as additive) and complementary information was obtained. nor-Harmane in the negative mode showed good results for detection of phosphorylated species. Furthermore, the use of this MALDI matrix in the negative-ion mode minimized the occurrence of prompt fragmentation giving good S/N ratio useful to perform LID-MS/MS. During MS/ MS measurements, preferential release of ester-bonded with respect to the amide-linked fatty acids occur, therefore determination of the acylation pattern was possible. This is very important since bacterial lipid A species are mixtures of similar molecules and the intensities of the spectra signals provide the relative proportions of the different species. In addition, in the positive-ion mode, nor-harmane allowed the detection of species bearing phosphoethanolamine groups. CMBT proved to be useful for the detection of phosphoethanolamine-containing species. Interestingly, with the addition of EDTA ammonium, not only the spectrum S/N ratio was increased but also the intensity of diphosphorylated species improved. Furthermore, signals attributed to lipid A species containing one phosphate and one phosphoethanolamine group were also detected. Notably, MALDI-MS of the lipid A moiety obtained from the minor phenol-extracted LPS using nor-harmane as matrix in the positive-ion mode exhibited the presence of ions in the high-mass range due to hepta-acylated species carrying two phosphoethanolamine groups that has not been previously observed in Sf LPS. Undoubtedly, the use of CMBT with EDTA ammonium as additive was essential to assure the presence of the hepta-acylated species. Interestingly, the intensity of the ions containing a phosphate and a phosphoethanolamine group was especially enhanced. In conclusion, MALDI-TOF-MS allowed us to unravel the lipid A heterogeneity, which was not previously reported in Sf LPS. It is well known that slight variations of the chemical structure of lipid A may change its biological activity. Thus, the knowledge of the detailed chemical structure represents an essential step for further development of new preventive or therapeutically active compounds.