Simplification of complex peptide mixtures for proteomic analysis: Reversible biotinylation of cysteinyl peptides

A rapid means of identifying many components in an enriched mixture of prot eins is enzymatic digestion of the entire protein fraction. This complex pe ptide mixture is then subjected to reversed-phase high performance liquid c hromatography (HPLC) coupled on-line with a mass spectrometer capable of da ta-dependent ion selection for fragmentation (LC-tandem mass spectrometry; MS/MS). Thus, as many peptides as possible in the sample are fragmented to produce MS/MS spectra, which can then be searched against sequence database s. Ideally, one peptide from each protein in the mixture would be fragmente d and identified. To this end, we employed an affinity selection method to capture cysteinyl peptides and thereby simplify the mixture. Both the captu red cysteinyl and the noncysteinyl peptides are analyzed by LC-MS/MS, to in crease the numer of proteins identified. The method was tested on a limited set of standard proteins and applied to the analysis of a protein fraction obtained from isolated mitochondria treated with atractyloside. To further increase the number of different precursor ions selected for fragmentation , dynamic exclusion and ion selection from multiple narrow mass ranges of c onsecutive runs were employed.