Analysis of Phosphorylated Peptides by Double Pseudoneutral Loss Extraction Coupled with Derivatization Using N-(4-Bromobenzoyl)aminoethanethiol

The analysis of post-translational phosphorylation is a crucial step in understanding the mechanisms of many physiological events. Numerous approaches for the development of analytical methods aimed at the detection and quantification of phosphorylated proteins by mass spectrometry have been reported in the literature. In this paper, we report a new strategy for the identification of phosphorylated serine and threonine residues in phosphoproteins. This method consists of selective derivatization of phosphoproteins coupled with double pseudoneutral loss extraction after nanoLC/ESI-MS/MS analysis. First, we designed and synthesized a new derivatization reagent, N-(4-bromobenzoyl)aminoethanethiol, which can selectively react with α,β-unsaturated ketones produced by β-elimination of a phosphoryl group from phosphorylated serine or threonine residues. The mass spectrum of the derivatized peptide shows a product ion with a characteristic isotopic pattern. After derivatization, fragment ions of peptides with pho...