1. Protein identification using MS/MS.
Protein identification using MS/MS. On the left is a depiction of the process in a typical 2D gel-based approach: the proteins are separated, visualized, and excised from the gel. On the right is the process involved for shotgun proteomics. In both approaches, sample proteins are proteolytically digested into peptides, and resulting peptides mixtures are separated using liquid chromatography. Peptides are ionized, and selected peptide ions are subjected to MS/MS sequencing. Peptide sequences are determined from MS/MS spectra using a database search approach. Any given peptide may be a part of the sequences of several different proteins. The protein inference problem involves figuring out which proteins are present in the sample given the sequences of identified peptides. In this example, the sample contains two proteins, A and B, which share extensive sequence homology. All three identified peptides, AEMK, GAGGLR, and HYFEDR, are present in the sequence of protein B, and the last two peptides are also in the sequence of protein A. In the shotgun proteomic approach, the connectivity between peptides and proteins is lost; no information on the number of proteins in the sample or their properties (e.g. molecular weight) is available. It is not possible to conclude that protein A is present in the sample because protein B can account for all observed peptides. This is less of a problem in the case of the 2D gel-based approach where proteins are separated prior to digestion and MS/MS analysis.
Alexey I. Nesvizhskii, and Ruedi Aebersold Mol Cell Proteomics 2005;4:
The American Society for Biochemistry and Molecular Biology