0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
nano spray ion source and by post source decay or TOF/TOF fragmentation,
in MALDI-TOF instruments.
This experimental setup would be as well extended to the relative quantitation
of the total protein content of subtypes of peripheral lymphocytes
(PBL) derived from human donors.
This research line will be followed in external collaboration with the
group of Luca Battistini, whose experience in the study of the role of T regulatory
cells (Treg) in autoimmune disorders and their state of the art technology
in the preparation and sub-fractionations of lymphocytes population will give
us a strong advantage. In particular Luca Battistini has identified CD39 as a
surface marker which unequivocally identifies T regulatory cells, and whose
expression confers unique functions.
They have also recently shown that CD39 is expressed by a fraction of
CD25bright cells, and that regulatory function is confined to this subset, while
the rest of CD25bright cells are highly activated conventional T cells, which
proliferate vigorously.
CD39 is a member of the family of ecto-nucleoside triphosphate diphosphohydrolases
(E-NTPase family) ectoenzymes, which metabolize ATP to 5’-
AMP, and it is the main enzyme involved in the regulation of extracellular
concentration of nucleotides. Extracellular ATP functions as a danger signal
released by damaged cells as chemo-attractant for lymphocytes, activator of
proinflammatory responses and inducer of local pain. The ectoenzyme CD39
removes the nucleotide by hydrolytic cleavage and therefore exhibits immediate
immune-modulatory influences on its proximal environment.
Notably, patients with the remitting/relapsing form of multiple sclerosis
(MS) have strikingly reduced numbers of CD39+ Treg cells in the blood. Thus,
in humans CD39 is a marker of a Treg subset likely involved in the control of
the inflammatory autoimmune disease.
In order to investigate on the differentially expressed proteins that potentially
confer suppressive abilities to the CD39+ subset, CD4+CD25bright
peripheral blood cells obtained from healthy individuals will be sorted based
on expression of CD39, and proteins expression from CD4+CD25brightCD39+
and CD4+CD25brightCD39- cells will be analysed by a shotgun proteomics
approach.
In brief extracts from different cell types will be trypsin digested and
injected in a nano Acquity UPLC coupled online with a nano ESI-Q-TOF mass
analyzer (Q-TOF Premiere, Micromass-Waters) working in “ MSe ” acquisition
mode. MSE data acquisition is a novel parallel peptide fragmentation protocol
that allows high peptide detection efficiency, ideal for protein identification
from very complex samples. Due to the high sensitivity and reproducibility
of the nanoLC system and the high-low characteristic collision energy
ramping of the mass spectrometer this approach is at the moment the most
promising for high throughput protein quantitation. The generated raw data
are then imported and processed by ProteinLynx Global Server (PLGS Micromass-Waters),
which provides proteins identification, relative quantitation
and statistical validation of results [Jeffrey et al. 2006].
Similar approach for metabolism investigation would be applied in the
630 2009