Reversed-phase liquid chromatography with double gradient elution for the separation and mass spectrometric analysis of peptides

Highly effective separation of complex peptide mixture is a prerequisite for protein identification with high coverage in proteomics. Currently, peptide mixture is separated by two-dimensional liquid chromatography (2D-LC) , ion exchange chromatography as the first dimension and reversed phase chromatography as the second dimension, in Shotgun proteomics. Though the 2D-LC is now widely used, its separation efficiency still needs further improvement. In this work, the first dimensional separation was performed by the pH and organic solvent double gradient elution. And then the two fractions, one from the early eluted section and the other from the later eluted section (with equal time intervals) were pooled and analyzed by MS/MS. The experimental results from the protein mixture of saccharomyces cerevisiae lysate showed that the separation by pH and organic solvent double-gradient elution, RP-HPLC-nanoRPLC coupled with MS/MS identified 567 more yeast protein groups (3 035 peptides) over strong cation exchange chromatography nanoRPLC coupled with MS/MS. The pI values and relative molecular masses of identified peptides ranged from 3.42 to 12. 01, and from 587. 67 to 3 499. 79, respectively. The pI values and relative molecular masses of identified proteins ranged from 3. 82 to 12. 19 and from 3 446. 55 to 432 905, respectively. These results indicated that this new 2DLC-MS method has the advantages over the conventional Shotgun method, and were expected to be applied in the separation of complex samples for proteomic studies in the future.