Enrichment strategy of cysteine-containing peptides based on covalent chromatography

Abstract

Automated multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) is routinely applied in large scale proteome profiling. However global proteome analysis remains a technical challenge due to the issues associated with sample complexity by tryptic digestion. The application of tag containing peptide enrichment approach for sample pre-separation could reduce the complexity of protein digest. Here, we demonstrated a simple and highly efficient cysteine-containing peptide enrichment method using a thiol specific covalent resin. The cysteine-containing peptides from the tryptic digests of the complex protein mixtures were selected by covalent chromatography based on thiol-disulfide exchange, identified by mass spectrometry. The strategy was firstly optimized and evaluated by using the tryptic peptides of bovine serum albumin (BSA). Then the method was applied with a relatively complicated sample from a five standard protein mixture. The results of these studies show that the enrichment method of cysteine-containing peptides is highly specific, efficient and reproducible. The effectiveness of this method in reducing the sample complexity and improving the identification of peptides by mass spectrometry has enabled high-throughput, automatic and large-scale qualitative and quantitative proteome analyses.