CIHIDECAR   12529
CENTRO DE INVESTIGACIONES EN HIDRATOS DE CARBONO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
134- MALDI-TOF-MS using different matrices is essential to unravel the lipid heterogeneity from bacterial lipopolysaccharides
Autor/es:
ADRIANA CASABUONO ; ALICIA S. COUTO
Lugar:
Los Cocos- Cordoba
Reunión:
Congreso; I Congreso Argentino de Espectrometría de masa; 2012
Institución organizadora:
SAEM
Resumen:
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 flexnerivariant 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 SfLPS. 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 SfLPS. 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.