Liquid chromatographic separation of proteins derivatized with a fluorogenic reagent at cysteinyl residues on a non-porous column for differential proteomics analysis

A wide-pore (30 nm) reversed-phase column (Intrada WP-RP, particle size 3 μm) was recently utilized for protein separation in differential proteomics analysis with fluorogenic derivatization-liquid chromatography–tandem mass spectrometry (FD–LC–MS/MS), and exerted a tremendous effect on finding biomarkers (e.g., for breast cancer). Further high-performance separation is required for highly complex protein mixtures. A recently prepared non-porous small-particle reversed-phase column (Presto FF-C18, particle size: 2 μm) was expected to more effectively separate derivatized protein mixtures than the wide-pore column. A preliminary experiment demonstrated that the peak capacity of the former was threefold greater than that of the latter in gradient elution of a fluorogenic derivatized model peptide, calcitonin. The FD–LC–MS/MS method with a non-porous column was then optimized and applied to separate liver mitochondrial proteins that were not efficiently separated with the wide-pore column. As a result, high-performance separation of mitochondrial proteins was accomplished, and differential proteomics analysis of liver mitochondrial proteins in a hepatitis-infected mouse model was achieved using the FD–LC–MS/MS method with the non-porous column. This result suggests the non-porous small-particle column as a replacement for the wide-pore column for differential proteomics analysis in the FD–LC–MS/MS method.