Automated Neutral Loss and Data Dependent Energy Resolved “Pseudo MS 3 ” for the Targeted Identification,Characterization and Quantitative Analysis of Methionine-Containing Peptides

Abstract

A strategy involving the fixed-charge sulfonium ion derivatization, stable isotope labeling, capillary high-performance liquid chromatography and automated data dependent neutral loss scan mode tandem mass spectrometry (MS/MS) and “pseudo multiple mass spectrometry (MS³)” product ion scans in a triple quadrupole mass spectrometer has been developed for the “targeted” gas-phase identification, characterization and quantitative analysis of low abundance methionine-containing peptides present within complex protein digests. Selective gas-phase “enrichment” and identification is performed via neutral loss scan mode MS/MS, by low energy collision-induced dissociation of the derivatized methionine side chain, resulting in the formation of a single characteristic product ion. Structural characterization of identified peptides is then achieved by automatically subjecting the characteristic neutral loss product ion to further dissociation by data dependent product ion scan mode pseudo MS³ under higher collision energy conditions. Quantitative analysis is achieved by measurement of the abundances of characteristic product ions formed by sequential neutral loss scan mode MS/MS experiments from “light” (¹²C) and “heavy” (¹³C) stable isotope encoded fixed-charge derivatized peptides. In contrast to MS-based quantitative analysis strategies, the neutral loss scan mode MS/MS method employed here was able to achieve accurate quantification for individual peptides at levels as low as 100fmol and at abundance ratios ranging from 0.1 to 10, present within a complex protein digest.

Keywords

peptide derivatization protein identification protein quantification tandem mass spectrometry proteomics

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