3. Protein analysis and identification

3.1 Enzymatic hydrolysis of proteins

This enzymatic hydrolysis step involves the controlled cutting of proteins into peptides. Proteins undergoing digestion may be in a liquid medium or embedded in an electrophoresis gel, which will be excised following protein migration and detection. Enzymatic hydrolysis is divided into three main stages.

• Denaturation/reduction breaks down the three-dimensional edifices and secondary structures that make up proteins, in particular intra- and interprotein disulfide bridges. It takes place in a buffered medium in the presence of chaotropic agents (5% trifluroethanol, 5/2 M urea/thiourea) and reducing agents (DTT, between 4 mM and 300 mM). When using urea/thiourea, it is essential to desalt the sample before mass spectrometric...