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Site‐specific separation and detection of phosphopeptide isomers with p H ‐mediated stacking capillary electrophoresis‐electrospray ionization‐tandem mass spectrometry
Author(s) -
Dong YuMing,
Chien KunYi,
Chen JengTing,
Lin ShihJie,
Wang TzuChien V.,
Yu JauSong
Publication year - 2013
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201300054
Subject(s) - phosphopeptide , chemistry , chromatography , tandem mass spectrometry , capillary electrophoresis–mass spectrometry , electrospray ionization , capillary electrophoresis , mass spectrometry , electrospray , stacking , protein mass spectrometry , extractive electrospray ionization , tandem , sample preparation in mass spectrometry , analytical chemistry (journal) , peptide , organic chemistry , materials science , composite material , biochemistry
This study reported a p H ‐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 T p PL 22 SPTR , SGAQASS 19 TPL 22 S p PTR , and SGAQASS 19 T p PL 22 S p P TR , 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.