Proteins are first cleaved into peptides by means of proteolytic enzymes such as trypsin, chymotrypsin or GluC. These peptides are separated by means of liquid chromatography and analyzed by means of mass spectrometry (LC-MS/MS). The spectra obtained by mass spectrometry are correlated to amino acid sequences, which on their turn are compared with databases from different kind of species (bacteria, human, mice, …).

At ProGenTomics, protein identification can be performed on a TripleTOF5600 (Sciex) or on a SynaptG2Si (Waters) by both data-dependent acquisition (DDA) and data-independent acquisition (DIA). DDA is currently the most often used strategy for protein identification where the most abundant precursors are selected for fragmentation. In DIA, on the contrary, all eluting peptides are fragmented which makes DIA an unbiased, reproducible quantitation method. 

By means of an in-house developed method, the identity of a biological matrix and the species of forensic samples can be determined as well. This approach has the advantage that no extra trace material is needed since the analysis is performed on the first “washing” step of the DNA-extraction, a solution which is normally discarded, and that one single test is sufficient to determine the identity and the species of the biological matrix, while the conventional methods require cascade testing (Van Steendam et al., 2012, Int J Legal Med.).

 

Advantages of DDA

  • Manual inspection of the spectra is possible
  • Gold standard for protein identification

 

Advantages of DIA

  • All peptides get fragmented
  • High reproducibility which is necessary for quantification

 

Workflow

You provide us with a protein sample, free from salts, preferably with a known amount. The proteins will first be digested into peptides by means of an enzyme. For quantification purposes, internal standards can be added to the sample. Peptides are first separated by means of hydrophobicity on a C18 column and subsequently analysed by a mass spectrometer (either TripleTOF 5600, Sciex or Synapt G2Si, Waters). The spectra will be searched against a database of choice and protein identification will be obtained after data-analysis with Mascot Daemon, Protein Pilot or ProteinLynx Global server (PLGS).

 

About ProGenTomics

ProGenTomics, located in Gent (Belgium), offers a wide range of proteomics and genomics services and support to the academic community and its external customers. We aim to tailor our services to the needs of our collaborators and customers, while giving great support.  

Contact us

ProGenTomics, proteomics & genomics services

  Ghent University, Faculty of Pharmaceutical Sciences

  Ottergemsesteenweg 460, 9000 Gent, Belgium

 

  +32 9 264 83 56