Coupling immobilized alkaline phosphatase-based automated diagonal capillary electrophoresis to tandem mass spectrometry for phosphopeptide analysis.

Automated diagonal capillary electrophoresis is a two-dimensional separation method that incorporates an immobilized enzyme reactor at the distal end of the first capillary and employs identical electrophoretic separation modes in both dimensions. Components undergo a preliminary separation in the first capillary. Fractions are parked in the reactor where some components undergo transformation. The fractions are then periodically transferred to the second capillary and replaced by the next components in the sample. Components that are not modified by the reactor will have identical mobility in both dimensions and fall on the diagonal of a reconstructed two-dimensional electropherogram, while analyte that undergoes modification will fall off the diagonal. In this study, alkaline phosphatase was immobilized in a monolithic reactor. An LTQ-Orbitrap Velos mass spectrometer was used to monitor analytes as they migrated from the second capillary. The system was used to characterize the phosphorylation status of a tryptic digest of α-casein in a background prepared from a 22-fold excess of the tryptic digest of bovine serum albumin. 120 fractions underwent automated treatment in the alkaline phosphatase reactor and separation in the second dimension capillary for over 40 min; nine phosphorylated α-casein peptides that produced 20 different phosphorylation states were detected with high confidence.