High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis

Orthogonal high-resolution separations are critical for attaining improved analytical dynamic range and protein coverage in proteomic measurements. High-pH reversed-phase liquid chromatography (RPLC), followed by fraction concatenation, affords better peptide analysis than conventional strong cation-exchange chromatography applied for 2D proteomic analysis. For example, concatenated high-pH RPLC increased identification of peptides (by 1.8-fold) and proteins (by 1.6-fold) in shotgun proteomics analyses of a digested human protein sample. Additional advantages of high-pH RPLC with fraction concatenation include improved protein sequence coverage, simplified sample processing and reduced sample losses, making this an attractive alternative to strong cation-exchange chromatography in conjunction with second-dimension low-pH RPLC for 2D proteomics analyses.