0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

FISM.9 – Proteomics and metabolomics investigations for the identification of multiple…
ried out by the use of exploratory data analysis processes, including basic statistics
and multivariate techniques designed to identify systematic relations
among variables and to cluster the different clinical groups. The statistical
analysis tools, actually in use in our laboratory, will be complemented by new
bioinformatics methods, as for example the Independent Component Analysis.
This is a signal processing technique that proved large reliability in the
extraction of protein signal profiles [Mantini et al. 2008].
At the end of data analysis, specific signals that are consistently found
within groups and are differently expressed across groups will be detected.
Signals which would specifically cluster with a clinical class would be thoroughly
characterised by means of further mass spectrometric investigations.
This procedure would employ the use of MALDI-TOF-MS reflectron
mode and nanoLC-TANDEM-MS on hybrid instruments quadrupole-time of
flight (Q-TOF Ultima, Waters-Micromass). The strategy follows a preliminary
chromatographic separation 1D or 2D, such as ionic exchanger coupled to a
reverse phase, in order to reduce sample complexity. Fractions containing the
signals of interest are then analysed by a mass finger print approach after proteolysis
with trypsin. The MALDI-TOF-MS reflectron mode and the nanoLC-
QTOF-MS/MS data obtained from these experiments would be then using to
fit genomic or protein databases, possibly returning unique protein identification.
These experiments might eventually lead to the development of a preliminary
diagnostic and/or prognostic assay for multiple sclerosis.
The investigation of protein patterns would be as well pursued by 2D-
PAGE analysis of CSF samples of patients pooled according to their clinical
profile and distribution of molecular markers from the linear MALDI-TOF-
MS analysis. 2D-PAGE protein profile analysis will be employed in conjunction
with MALDI-TOF – MS for the analysis of tryptic digests for mass finger
print identification. Each analysed sample would be replicated in 2D-PAGE
independent experiments and the obtained average master differentially evaluated.
Differences in spot volumes greater then 5 fold increment or decrement
would be considered significant.
Mass finger print experiment will be acquired on a MALDI-TOF Reflex IV
or Ultraflex III instruments (Bruker-Daltonics) operating in reflectron/positive
ion mode. Within the investigation of protein post-translational modifications
we will focused our attention on protein carbonylation. These investigation
will take advantage from “ in strip derivatization technique ” including internal
standard thus enabling to perform a relative quantification of carbonylated
proteins in the CSF. Samples are separated on non-linear 3-10 pH range
1 st dimension and after focusing IPG strips are derivatized with 2,4 dinitrophenylhydrazine
(DNPH). Carbonylated proteins are detected using anti-DNP
antibody. This method allows the visualization of oxidized proteins by 2-DE
with high reproducibility because the derivatization is made after the focusing
step avoiding pI alterations.
The fine micro-characterization of protein primary structure and post
translation modifications would be pursued via fragmentation analysis of proteolytic
peptides. In order to perform these experiments we have developed
MS/MS experiments both on hybrid ESI-Q-TOF instrument, equipped with a
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