Identification

Generally, proteins of interest are separated by gel electrophoresis and subjected to a controlled enzymatic digestion using an endoprotease. Due to its high specificity, the most commonly used enzyme is trypsin. Trypsin cleaves the proteins after the Lysine and Arginine residues. Enzymatic digestion of proteins by the trypsine leads generally leads to the formation of peptides of 800 to 3000 Da.

Then, the mass of the different peptides generated by the digestion of a protein is precisely measured using a MALDI-TOF or a MALDI-TOF-TOF mass spectrometer. This list of masses constitutes a specific fingerprint of the protein and is exported in search engines dedicated to proteomic analysis to perform the interrogation of sequence databases.

These softwares perform the theoretical digestion of all protein sequences of the databases (including protein sequences resulting from the translation of nuceotidic sequences) and compare compare the experimental mass list to the theoretical mass list. A protein is identified when a sufficient number of correct correlations (number of peptides whose experimetal mass correponds to the theoretical mass, mass accuracy, protein sequence coverage, etc...) is found.

This technique is used for the identification of an abundant protein in a sample of low complexity (2D gel spots or visible band of 1D gel) but is not appicable to the identification of proteins in complex samples.

Analysis by tandem mass spectrometry (MS/MS) provides information on the amino acids sequence of the peptides resulting from tryptic digestion and can be used if no identification has been obtain using the peptide mass fingerprint method or to identify several proteins in a complex mixture.

The proteolytic peptides are ionized by ESI (electrospray ionization) or MALDI (matrix-assisted laser desorption ionization), separated by an analyzer and detected to generate a MS spectrum. In a MS/MS experiment, an ion of particular m/z (mass to charge) ratio, named precursor ion, is selected and fragmentated into fragment ions. These fragment ions are then selected and detected to generate a MS/MS spectrum.

Les pics de masse obtenus constituent une représentation de la séquence protéique, dans laquelle deux pics adjacents diffèrent par la masse d’un acide aminé perdu lors de la fragmentation du peptide analysé. L'analyse approfondie de l'enchainement de ces pics permet de déterminer une partie de la séquence de l'ion parent et donc d'identifier la protéine de départ.

The masses of the ions observed on the MS/MS spectrum can provide information on the protein sequence. Indeed, when an amino acid is lost during MS/MS fragmentation of the selected peptide, two peaks presenting a difference of mass corresponding to the mass of this amino acid are observed. The analysis of the serie of peaks allows the determination of a a part of the amino acid sequence of the peptide that can provide the identification of the protein.

This technique is used to identify proteins in a complex mixture or for the validation of identifications obtained by peptide mass fingerprint.