Thiophosphorylation and Fluorescent Labeling of Substrate Peptide of Protein Kinase

Study on the conditions of thiophosphorylation reaction and fluorescent labeling reaction of the substrate of protein kinase A was carried out by using Kemptide LRRASLG as a model. The suitable concentration of the fluorescent regent 5-{ ((2-iodoacetyl)amino)ethyl amino} naphthelene-1-sulfonic acid (1,5-IAEDANS) and the suitable pH of labeling reaction buffer were 1.6 mmol/L and 8, respectively. Stability of the labeled peptide under the conditions of automatic N-terminal protein sequencing, electrospray mass spectrometry and in the presence of 0.1% trifluoroacetic acid was investigated, respectively. The specific differences in UV spectra between the labeled and unlabeled peptides were observed. Therefore, the possibility to detect the thiophosphorylated and fluorophore labeled peptide during high performance liquid chromatography peptide mapping was primarily shown.