Peptide mass mapping constrained with stable isotope-tagged peptides for identification of protein mixtures

Through proteolysis and peptide mass determination using mass spectrometry, a peptide mass map (PI IM) can be generated for protein identification. Ho wever, insufficient peptide mass accuracy and protein sequence coverage lim it the potential of the PMM approach for high-throughput, large-scale analy sis of proteins. In our novel approach, nonlabile protons in particular ami no acid residues were replaced with deuteriums to mass-tag proteins of the S. cerevisiae proteome in a sequence-specific manner. The resulting mass-ta gged proteolytic peptides with characteristic mass-split patterns can be id entified in the data search using constraints of both amino acid compositio n and mass-to-charge ratio. More importantly, the mass-tagged peptides can further act as internal calibrants with high confidence in a PMM to identif y the parent proteins at modest mass accuracy and low sequence coverage. As a result, the specificity and accuracy of a PMM was greatly enhanced witho ut the need for peptide sequencing or instrumental improvements to obtain i ncreased mass accuracy. The power of PMM has been extended to the unambiguo us identification of multiple proteins in a ID SDS gel band including the i dentification of a membrane protein.