Single shot CZE-MS/MS for phosphoproteomics

The dependence of capillary zone electrophoresis separations on the charge state of analyte is useful for the analysis of many post-translational modifications in proteins. In this work, we coupled CZE to an Orbitrap Fusion Lumos Tribrid platform with advanced peak determination algorithm for phosphoproteomics analysis. A linear polyacrylamide coated capillary with very low electroosmotic flow was used for the separation. The optimal injection volume is between 100 nL and 150 nL of phosphopeptides dissolved in 30 mM ammonium bicarbonate (pH 8.2) buffer, which produces a dynamic pH junction sample injection. Larger injection volumes resulted in serious peak broadening and decreased numbers of phosphopeptide identifications. The optimized system identified 4405 phosphopeptides from 220 ng of enriched phosphopeptides from mouse brain, which represents the state-of-the-art result for single shot CZE-ESI-MS/MS based phosphoproteome analysis.