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Liquid-based
approaches
In the
liquid based proteomics, the identification and quantification of peptides and
proteins is performed as well as identification of post-translational
modifications, de novo sequencing and quantification and
identification of metabolites. For these multiple alternative services
the equipment used is a comprehensive 2D-HPLC (Ultimate 3000) coupled to
an ESI source (nano or Turbo V) and hybrid triple quadrupole/linear ion
trap (QTRAP 4000) mass spectrometer and/or a 2D-nanoLC Ultra (Eksigent)
coupled to the 5600 Triple TOF (ABsciex)
For
identification of proteins, one-dimension reverse phase nanoLC-ESI-MS/MS
is currently being used. Proteins from gel (1D, 2D) or in solution are
digested (normally, with trypsin) and their peptides are injected into
the system where they are separated according to their hidrophobicity
until they reach the mass spectrometer.
Peptides
resulted from digestion of complex mixtures of proteins are separated by
two-dimensional liquid chromatography. They are separated in the first
dimension by strong cation exchange, resulting in different fractions
from different salt concentration. The content of each fraction is then
separated in the second dimension by reverse phase chromatography. As
for one-dimension liquid chromatography referred above, individual
peptides undergo induced fragmentation and yield MS/MS spectra.
Proteins
are identified by submitting all MS/MS spectra to a database, as
SwissProt, with different softwares being used for the search: Mascot,
Protein Pilot or Peaks, based in different search algorithms.
For
identification and location of post-translational modifications (phosphorylations,
glycosylations) a proteolytic digestion is performed and modifications
are detected by the mass spectrometer using different acquisition modes
(Neutral Loss, Precursor Ion Scan or Multiple Reaction Monitoring - MRM
modes). ProteinPilot software enables the searching for hundreds of
peptide modifications and is normally used for this type of
identification.
Quantification of metabolites is achieved by using the mass spectrometer
in MRM mode, where the target ion is selected in the first quadrupole,
fragmented in the second quadrupole, and just one fragment is selected
in the last quadrupole to reach the detector. MRM mode permits an
increase in sensitivity as well as specificity. In the same way,
quantification of peptides and proteins can be achieved using the
Multiquant software.
We also
use quantification based on labelling tags as iTRAQ, where a multiplexed set of
two to eight isobaric amine specific reagents yield differently labelled
peptides. This allows simultaneous identification and quantification of
proteins from different conditions in one single experiment.
Finally, label
free quantification can be performed on intact ions and also in
fragments using the recently developed SWATH data acquisition method and
processing using both PEAKView and MarkerView
-4000 QTRAP mass spectrometer
was acquired with founds from "Programa Nacional de Reequipamento
Cientifico"
and the CNC is
member of the Portuguese National Mass Spectrometry Network
-Triple TOF5600 - Eksigent nanoLC, A aquisicao destes equipamentos foi financiada atraves do QREN - Programa Operacional Regional do Centro 2007-2013 com o apoio do Mais Centro e da Uniao Europeia no
ambito do projeto "CNC Biotech - investigacao em Biotecnologia e capacitacao do sector empresarial"
-Triple
TOF6600 mass spectrometer
was acquired with founds from ROTEIRO/0028/2013
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