title: Site-specific separation and detection of phosphopeptide isomers with pH-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.
authors.affiliation: School of Pharmacy, Lanzhou University, PR China.
authors.forename: Yu-Ming
authors.initials: YM
authors.lastname: Dong
authors.forename: Kun-Yi
authors.initials: KY
authors.lastname: Chien
authors.forename: Jeng-Ting
authors.initials: JT
authors.lastname: Chen
authors.forename: Shih-Jie
authors.initials: SJ
authors.lastname: Lin
authors.forename: Tzu-Chien V
authors.initials: TC
authors.lastname: Wang
authors.forename: Jau-Song
authors.initials: JS
authors.lastname: Yu
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007536:Isomerism; D034882:Lamins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D034882:Lamins; D010748:Phosphopeptides; D005982:Glutathione Transferase; D016203:CDC2 Protein Kinase