Site‐specific separation and detection of phosphopeptide isomers with p<scp>H</scp>‐mediated stacking capillary electrophoresis‐electrospray ionization‐tandem mass spectrometry

Abstract

This study reported a pH-mediated stacking CE coupled with ESI MS/MS method to determine the phosphorylation sites of three synthetic phosphopeptides containing structural isomers. These phosphopeptides mimic the phosphopeptides (amino acid residues 12-25) derived from the trypsin-digested products of human lamin A/C protein. The LODs were determined to be 118, 132 and 1240 fmol for SGAQASS(19)TpPL(22)SPTR, SGAQASS(19)TPL(22)SpPTR, and SGAQASS(19)TpPL(22)SpPTR, respectively. The established method was employed to analyze the phosphorylation sites of the trypsin-digested products of glutathione S-transferase-lamin A/C (1-57) fusion protein that had been phosphorylated in vitro by cyclin-dependent kinase 1. The results indicated that this method is feasible to specifically determine the phosphorylation site from phosphopeptide isomers in the trypsin-digested products of a kinase-catalyzed phosphoprotein, which should benefit the investigation of protein kinase-mediated cellular signal transduction.