Site‐specific separation and detection of phosphopeptide isomers with pH‐mediated stacking capillary electrophoresis‐electrospray ionization‐tandem mass spectrometry

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 SGAQASS¹⁹TpPL²²SPTR, SGAQASS¹⁹TPL²²SpPTR, and SGAQASS¹⁹TpPL²²SpPTR, 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.