Development of an online two-dimensional nano-scale liquid chromatography/mass spectrometry method for improved chromatographic performance and hydrophobic peptide recovery

An online two-dimensional (2D) strong cation-exchange (SCX)/reversed-phase (RP) nano-scale liquid chromatography/mass spectrometry (nanoLC/MS) method was developed for improved separation and hydrophobic peptide recovery. Sharper and more symmetric RP peaks were observed with the use of a “band re-focusing method”, in which an analytical RP column with more hydrophobicity than the RP trap column was used in the system. To recover hydrophobic peptides still unreleased from the SCX column after a conventional salt step gradient due to hydrophobic interaction, a RP step gradient from 10% to 30% acetonitrile (ACN) was applied to the SCX column in the presence of a high salt concentration following the salt gradient. There were 301 unique hydrophobic E. coli peptides identified from the RP fractions. These peptides, which were 19% of all E. coli peptides identified from a 2D run, would not have been identified without the application of the RP gradient to the SCX column.