Preparation of capillary hybrid monolithic column with sulfonate strong cation exchanger for proteome analysis

Strong cation exchange (SCX) chromatography is one of the most important separation modes in liquid chromatography and SCX column is widely applied in high resolution separation or fractionation of various samples. In this work, a sulfonate SCX hybrid monolithic column was successfully prepared by “one-pot” strategy and the hybrid monolith is well optimized to obtain homogenous structure. It was observed that this sulfonate SCX hybrid monolithic column had ∼7 times permeability (in water) and ∼3 times sample loading capacity (tested by dipeptide Gly-Tyr) comparing to particulate SCX column packed with commercial available material. Then, it was used as trap column for fast sample loading of the enriched phosphopeptides. Comparing to phosphate SCX polymer monolithic column, the number of identified phosphopeptides increased ∼19% due to the sulfonate group has higher retention strength than phosphate group for peptide cations. And the coverage of phosphoproteome obtained by sulfonate SCX hybrid monolithic column is similar to particulate packed SCX column, because they had identical sulfonate group to retain the peptide cations. Finally, the sulfonate SCX hybrid monolithic column was used as enzyme reactor for online protein digestion. Comparing to particulate SCX packed column, the identified peptides number increased 40% and the protein coverage increased 10%. This might be ascribed to the high porous structure and relative high surface area that elevated the digestion efficiency.