A new fluorescent quenching method for the determination of phosphoproteins by using calconcarboxylic acid

A fluorescent quenching detection method for phosphoproteins in SDS-PAGE by using calconcarboxylic acid (CCA) was described. In this method, the fluorescence intensity of CCA was greatly increased with the presence of Al(3+) in the gel background, while in zones where phosphoproteins are located this intensity was absent because of fluorescence quenching phenomenon through the formation of CCA-Al(3+) -phosphoprotein appended complex. Approximately 4-8 ng of phosphoproteins can be selectively detected within 1 h (1D SDS-PAGE), which is similar to that of the most commonly used Pro-Q Diamond stain. The specificity of this novel technique for phosphoproteins was confirmed by dephosphorylation, Western blot, and LC-MS/MS analysis, respectively. Furthermore, to better understand the newly developed method, the detection mechanism of CCA stain was explored by fluorescent spectrometry. According to the results, it is believed that CCA stain may provide a new choice for selective, economical, MS compatible, and convenient visualization of gel-separated phosphoproteins.