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 phosphoserine or phosphothreonine have twin peaks with an intensity ratio of approximately 1:1 and a difference of two mass units, while product ions from peptides without phosphoserine or phosphothreonine have normal isotopic patterns. Therefore, the neutral loss of the derivatized residue in the product ion mass spectrum includes two difference losses caused by ⁷⁹Br and ⁸¹Br. The extraction of the product scan mass spectrum with double pseudoneutral losses is very effective for identification of the derivatized peptides produced from phosphorylated peptides. The new strategy represents a useful tool for the analysis of phosphorylated serine and threonine residues in phosphorylated proteins.