Continuous pH/salt gradient and peptide score for strong cation exchange chromatography in 2D-nano-LC/MS/MS peptide identification for proteomics.

Tryptic digests of human serum albumin and human lung epithelial cell lysates were used as test samples in a novel proteomics study. Peptides were separated and analyzed using 2D-nano-LC/MS/MS with strong cation exchange (SCX) and reversed-phase chromatography and continuous gradient elution. The peptide elution conditions combined simultaneous pH gradient with ammonium acetate salt gradient elution modes. A novel empirical SCX peptide elution score was developed, which accounts for both the number of basic and acidic residues and, in part, their location within a sequence of a peptide. Average scores calculated for the fractionated peptide sequences correlated well with the pH of SCX elution fractions. Multiple peptides with identical amino acid sequences, but differing in cysteine tags possessing different positive charge and different SCX elution properties, were obtained by subjecting the samples to reduction and alkylation with different cysteine alkylating reagents: iodoacetamide, 4-vinylpyridine, and (3-acrylamidopropyl) trimethylammonium chloride. The structurally similar peptides were used as elution standards.