Whole-cell protein identification using the concept of unique peptides

A concept of unique peptides (CUP) was proposed and implemented to identify whole-cell proteins from tandem mass spectrometry (MS/MS) ion spectra. A unique peptide is defined as a peptide, irrespective of its length, that exists only in one protein of a proteome of interest, despite the fact that this peptide may appear more than once in the same protein. Integrating CUP, a two-step whole-cell protein identification strategy was developed to further increase the confidence of identified proteins. A dataset containing 40,243 MS/MS ion spectra of Saccharomyces cerevisiae and protein identification tools including Mascot and SEQUEST were used to illustrate the proposed concept and strategy. Without implementing CUP, the proteins identified by SEQUEST are 2.26 fold of those identified by Mascot. When CUP was applied, the proteins bearing unique peptides identified by SEQUEST are 3.89 fold of those identified by Mascot. By cross-comparing two sets of identified proteins, only 89 common proteins derived from CUP were found. The key discrepancy between identified proteins was resulted from the filtering criteria employed by each protein identification tool. According to the origin of peptides classified by CUP and the commonality of proteins recognized by protein identification tools, all identified proteins were cross-compared, resulting in four groups of proteins possessing different levels of assigned confidence.