Protein Identification by SEQUEST

Abstract

The idea that a set of (poly)peptide masses obtained by specific enzymatic or chemical cleavage can be used as a unique fingerprint, allowing a protein to be identified in a database, was first proposed in 1977 (Cleveland et al 1977). The masses of the polypeptides generated by in-gel proteolysis (estimated from sodium dodecyl sulphate polyacrylamide-gel electrophoresis gels) were used to identify viral-coat proteins. The mass accuracy obtained by gel electrophoresis was sufficient to allow identification, because the size of the database was so small. Later, the combination of peptide mapping and mass spectrometry (MS) was introduced as a method for verifying and/or correcting the primary structures of proteins, as deduced from their corresponding gene sequence (Gibson and Biemann 1984; Morris et al. 1984; Greer et al. 1988). The strategy implied the use of fast atom bombardment (FAB) or plasma-desorption MS to determine the masses of the proteolytic fragments produced by a specific enzyme or chemical. The experimental data was then compared with the masses predicted from all three reading frames of the gene sequences in order to verify the protein sequence or show the presence of post-translational modifications. Greer demonstrated that FAB mapping allowed the confirmation of 93% of the sequence of recombinant 1-anti-trypsin molecules (Greer et al. 1988); this introduced the use of MS-based quality control of expressed proteins.