Searching sequence databases over the internet: protein identification using MS-tag

Although peptide mass mapping is fast and simple, its success can be compromised by the purity of the protein in one gel spot, errors in the sequence database, and the detection of too few peptides in the MS map of a given protein to permit reliable database matching. In those cases, partial amino acid sequence that is determined using post-source decay (PSD) analysis or tandem mass spectrometry is required to establish the correct protein identification among possibilities suggested by MS-Fit. Post-source decay (PSD) involves the detection of the fragmentation product ions (metastable ions) of a selected mass value window containing the precursor ion chosen, that occur in the field-free region between the ion source and the reflectron. This unit covers some of the practical issues in obtaining and analyzing a typical PSD spectrum using MS-Tag, a database interrogation program that enables the identification of proteins with a limited number of peptide fragment ions by matching existing database sequences. It is a user-friendly program that simultaneously considers a variety of ion types, but still allows single-mismatch-tolerant searching. Although MALDI-PSD database searching is a widely used tool, an alternative method also described in this unit is analysis by MALDI collision-induced dissociation (MALDI-CID).