Label-free electrochemical detection of the phosphorylated and non-phosphorylated forms of peptides based on tyrosine oxidation

Abstract

Identification of protein phosphorylation is an important goal in proteomics, because of the central role played by phosphorylation in the regulation of cellular activities. An exciting consequence of tyrosine (Tyr) oxidation is that it allows a clear distinction between the phosphorylated and non-phosphorylated forms of peptides using electrochemical analysis. In this report, we monitored the effect of phosphorylation on the electro-oxidation of Tyr in connection with differential pulse voltammetry (DPV) using a screen-printed carbon electrode (SPCE). First, we monitored the electrochemical current responses of Tyr and o-phospho- l-Tyrosine ( l-3-(4-hydroxyphenyl)alanine 4′-phosphate, Tyr-P). The detection limit for Tyr was determined as 10 nM on the SPCE surface ( S/ N = 3). We observed that the phosphorylation caused a significant suppression on the electro-oxidation of Tyr. We also monitored the electrochemical responses of Src peptide 521–533 (H–Thr-Ser-Thr-Glu-Pro-Gln-Tyr-Gln-Pro-Gly-Glu-Asn-Leu–OH), both in the non-phosphorylated and phosphorylated forms. The detection limit for Src peptide was determined as 100 nM ( S/ N = 3). By monitoring the current signals obtained from the Tyr kinase substrate peptides, we suggest that label-free electrochemical in vitro detection of Tyr phosphorylation can be performed in a rapid and cost-effective format.