0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
– Control group, remitting MS in resting phase, relapsing MS in acute
phase, secondary- progressive MS, primary-progressive MS.
We shall take particular care in selecting on a separate group patients
newly diagnosed and not under pharmacological treatment at the time of
lumbar puncture. Patients will be evaluated in their degree of functional
disability following the Extended Disability Status Scale (EDSS) and by the
MMSE (Mini Mental Status Exam) to get information on potential cognitive
deficits. All patients affected by multiple sclerosis would be investigated
in their CSF oligoclonal components of immunoglobulin G by
immuno isoelectrofocalization assays. Blood-brain barrier index would be
evaluated. The level of brain atrophy would be, as well, evaluated by magnetic
resonance.
The control groups would include healthy controls and patients with the
following neurological disturbances: lower extremity radiculopathy, polyneuropathy,
Guillain-Barré. During the time loan of this proposal we plan to
investigate a minimal number of 100 patients divided between the different
classes of investigation.
The proposed research would employ a large use of mass spectrometry
coupled to bio-informatics analysis on genomic and/or protein databases. In
our laboratory we have developed a number of state of the art approaches in
mass spectrometry analysis of biological samples. These techniques are taking
full advantage of the available mass spectrometry instrumentations: HPLC-
ESI Triple Quadruple MS, nanoHPLC-ESI Q-TOF MS, GC-MS and MS/MS,
MALDI-TOF-MS and MS/MS.
The proteomics investigation will be initially pursued by the use of linear
MALDI-TOF-MS assay to obtain fast and reproducible protein profiles of CSF
and serum samples of the enrolled patients. This technique would allow an initial
screening of the 2 different low molecular weight regions polypeptide (800-
5000 Da, with oxidized insulin as internal standard and a second range 5000-
20000 Da with horse myoglobin as internal standard). The sample preparation
and MALDI ionization conditions would be optimised by an open matrix factorial
screening in order to match experimental conditions, which might discriminate
the different clinical groups enrolled. This initial optimisation would
be pursued by the use of different sample extraction methodologies. Our linear
MALDI-TOF-MS method applied in a preliminary investigation of CSF and
serum samples in positive ion mode shows good analytical performance with a
FWHM resolution higher than 1500 at 15,000 Th and a mass accuracy of +/- 1
Th after internal calibration on haemoglobin and internal standard signals
(M+H+ and M+2H+). Further optimisation would be pursued in order to maximise
the number of discriminating signals.
Data will be collected on a Reflex IV and on a Ultraflex III MALDI-TOF-
MS and MS/MS in positive ion mode (Bruker-Daltonics). Mass spectra will be
processed by means of commercial packages and LIMPIC, an in-house developed
software.
The latter is a computational method for data denoising and peak detection.
A complete statistical analysis of mass spectrometer signals will be car-
628 2009