SCX charge state selective separation of tryptic peptides combined with 2D-RP-HPLC allows for detailed proteome mapping

• Published in: Journal of proteomics 2013-10, Vol.91, p.164-171
• Main Authors: Betancourt, Lázaro H., De Bock, Pieter-Jan, Staes, An, Timmerman, Evy, Perez-Riverol, Yasset, Sanchez, Aniel, Besada, Vladimir, Gonzalez, Luis Javier, Vandekerckhove, Joël, Gevaert, Kris
• Format: Article
• Language: English
• Subjects: amino acid composition; buffers; Cation exchange; fibroblasts; fractionation; mice; peptides; proteins; proteome; Proteomics; reversed-phase liquid chromatography; Reversible alpha-amino group and lysine modification; Selective peptide isolation
• ISSN: 1874-3919
• DOI: 10.1016/j.jprot.2013.06.033

Multidimensional peptide fractionation is widely used in proteomics to reduce the complexity of peptide mixtures prior to mass spectrometric analysis. Here, we describe the sequential use of strong cation exchange and reversed phase liquid chromatography in both basic and acidic pH buffers for separating tryptic peptides from complex mixtures of proteins. Strong cation exchange exclusively separates peptide by their charge state into neutral, singly and multi-charged species. To further reduce complexity, each peptide group was separated by reversed phase liquid chromatography at basic pH and the resultant fractions were analyzed by LC–MS/MS. This workflow was applied to a soluble protein lysate from mouse embryonic fibroblast cells, and more than 5000 proteins from 29,843 peptides were identified. The high selectivity displayed during the SCX step (93% to 100%) and the overlaps between proteins identified from the SCX-separated peptide groups, are interesting assets of the procedure. The present work shows how complex mixture of peptides can be selectively separated by SCX based essentially on the net charge of peptides. The proposed workflow results in three well-defined subset of peptides of specific amino acid composition, which are representative of the constituent proteins. The very high selectivity obtained (93% to 99%) on the peptide side, underscores for the first time the possibility of SCX chromatography to aid in validating identified peptides. [Display omitted] •SCX exclusively separates tryptic peptides by their charge state.•0, 1+ and multi-charged peptides are isolated with 93–99% of selectivity.•These subsets are further fractionated by 2D-RP-HPLC.•Detailed proteome mapping is achieved.•SCX could be used as a powerful tool for validating peptide identifications.