Double pseudo-neutral loss extraction coupled with specific derivatization for analysis of phosphoproteins

摘要

In this paper, we described the development of a new derivatization reagent, N-(4-bromobenzoyl)aminoethanethiol, useful for the selective analysis of phosphorylated proteins. This reagent reacts with alpha,beta-unsaturated ketones produced by the beta-elimination of phosphoryl groups from phosphorylated serine and threonine residues. The product ion mass spectra of the derivatized thioether products possess a characteristic isotopic pattern in which the derivatized residue-containing fragment ions have twin peaks with an intensity ratio of approximately 1:1 and a difference of two mass units. These characteristics permit the effective identification of phosphorylated serine and threonine residues in peptides. Moreover, derivatized peptides can be detected in a complicated peptide mixture by use of the double pseudo-neutral loss extraction method and analysis of the extracted peaks for the presence of the characteristic isotopic pattern. The novel analytical method will be useful for the identification of phosphorylated serine and threonine residues in proteins.