Label-free electrochemical differentiation of phosphorylated and non-phosphorylated peptide by electro-catalyzed tyrosine oxidation.
Protein phosphorylation plays an important role in many significant cellular processes, and has thus gained tremendous interest in the field of proteomics. The electro-active tyrosine residue, as an important receptor of phosphorylation in proteins, exhibits electro-inactivity after being phosphorylated on the hydroxy group of its aromatic ring. In this study, the electrochemical oxidation of tyrosine on indium tin oxide (ITO) electrodes was catalyzed with an electron mediator Os(bpy)(3)(2+) (bpy = 2,2'-bipyridine) and was employed as a signal reporter to differentially detect non-phosphorylated and phosphorylated peptides. A short, tyrosine-containing peptide glu-glu-glu-glu-glu-tyr (EY-6) was immobilized on an ITO surface using the layer-by-layer self-assembly method, and was detected by cyclic voltammetry in an Os(bpy)(3)(2+) solution. The limit of detection was about 0.23 microg mL(-1) EY-6 in solution. The phosphorylated peptide glu-glu-glu-glu-glu-tyr-OP (EY-6P) did not produce an appreciable oxidation current on the electrode. Surface plasmon resonance measurements revealed that the amount of EY-6 and EY-6P adsorbed on the sensor chip surface was 269 and 378 pg mm(-2), respectively. The poly(glu, tyr) (4 : 1) peptide, a protein tyrosine kinase substrate, was also detected by the same approach, with a detection limit of 0.65 microg mL(-1). This new approach offers the possibility of label-free and on-chip detection of protein tyrosine kinase activity.
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State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085, China.
Wan
Bin
B
Guo
Liang-Hong
LH
D000818:Animals; D002384:Catalysis; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D017971:Tin Compounds / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D010455:Peptides; D010748:Phosphopeptides; D017971:Tin Compounds; D014443:Tyrosine; C109984:indium tin oxide