Two-Dimensional Reversed-Phase × Ion-Pair Reversed-Phase HPLC: An Alternative Approach to High-Resolution Peptide Separation for Shotgun Proteome Analysis

A two-dimensional separation scheme for shotgun proteome analysis employing high-pH reversed-phase HPLC in the first and low-pH ion-pair reversed-phase HPLC in the second dimension (RP × IP-RP-HPLC) has been developed and evaluated. Compared to the classical strong cation exchange × ion-pair reversed-phase (SCX × IP-RP-HPLC) approach, the RP × IP-RP-HPLC system was characterized by a lower degree of orthogonality, which was, however, more than counterbalanced by higher separation efficiency, more homogeneous distribution of peptide elution, and easier experimental handling. Peptide fragment fingerprinting by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) was employed for peptide detection and identification. About 13% more peptides and 7% more proteins could be identified with the alternative approach in 30% less analysis time, enabling the analysis of the proteome of Corynebacterium glutamicum with a coverage of 24.9% (745 proteins). Combining the identification results both of the SCX- × IP-RP-HPLC-ESI-MS/MS and RP- × IP-RP-HPLC-ESI-MS/MS methods, a total of 871 proteins were identified in a cytosolic protein preparation, which represented 29.1% of all proteins annotated in the genome of C. glutamicum. Keywords: shotgun proteome analysis • multidimensional separation • high-pH reversed-phase chromatography • monolithic columns • low-pH ion-pair reversed-phase chromatography tandem mass spectrometry • Corynebacterium glutamicum