Preparation of linear polyacrylamide coating and strong cationic exchange hybrid monolith in a single capillary, and its application as an automated platform for bottom-up proteomics by capillary electrophoresis-mass spectrometry

Abstract

A linear polyacrylamide (LPA) coating and a sulfonate-silica hybrid strong cation exchange (SCX) monolith were prepared sequentially in a single fused silica capillary. The SCX-LPA capillary was used for sample preconcentration using solid-phase extraction (SPE) followed by capillary zone electrophoresis (CZE) separation and mass spectrometry detection. Samples were prepared in 1 M acetic acid, loaded by pressure onto the SCX SPE monolith, and eluted using 200 mM ammonium bicarbonate (pH 8). The background electrolyte for CZE was 1 M acetic acid. This combination of elution buffer and background electrolyte results in formation of a dynamic pH junction, which further improves the preconcentration performance. All experiments were performed using a CZE autosampler and an electrokinetically pumped nanospray interface. By loading 50 ng of Xenopus laevis eggs protein digest, 330 protein groups and 872 peptides were identified. The system was also applied to the analysis of 11 μL of a 10−4 mg⋅mL−1 (1.1 ng) bovine serum albumin tryptic digest; 12 peptides were identified and the protein coverage was 25%. Finally, by loading 5.5 μL of a 10−3 mg⋅mL−1 (5.5 ng) E. coli digest, 145 protein groups and 365 peptides were identified.