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Liquid-based proteomics

 

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

 

 

   
 
 
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