Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization

Abstract

An amine specific peptide derivatization strategy involving the use of novel isobaric stable isotope encoded ‘fixed charge’ sulfonium ion reagents, coupled with an analysis strategy employing capillary HPLC, ESI-MS, and automated data dependent ion trap CID-MS/MS, -MS3, and/or ETD-MS/MS, has been developed for the improved quantitative analysis of protein phosphorylation, and for identification and characterization of their site(s) of modification. Derivatization of 50 synthetic phosphopeptides with S,S′-dimethylthiobutanoylhydroxysuccinimide ester iodide (DMBNHS), followed by analysis using capillary HPLC-ESI-MS, yielded an average 2.5-fold increase in ionization efficiencies and a significant increase in the presence and/or abundance of higher charge state precursor ions compared to the non-derivatized phosphopeptides. Notably, 44% of the phosphopeptides (22 of 50) in their underivatized states yielded precursor ions whose maximum charge states corresponded to +2, while only 8% (4 of 50) remained at this maximum charge state following DMBNHS derivatization. Quantitative analysis was achieved by measuring the abundances of the diagnostic product ions corresponding to the neutral losses of ‘light’ (S(CH3)2) and ‘heavy’ (S(CD3)2) dimethylsulfide exclusively formed upon CID-MS/MS of isobaric stable isotope labeled forms of the DMBNHS derivatized phosphopeptides. Under these conditions, the phosphate group stayed intact. Access for a greater number of peptides to provide enhanced phosphopeptide sequence identification and phosphorylation site characterization was achieved via automated data-dependent CID-MS3 or ETD-MS/MS analysis due to the formation of the higher charge state precursor ions. Importantly, improved sequence coverage was observed using ETD-MS/MS following introduction of the sulfonium ion fixed charge, but with no detrimental effects on ETD fragmentation efficiency.

Peptides derivatized with bicyclic quaternary ammonium ionization tags. Sequencing via tandem mass spectrometry

“…Increased chromatographic retention was observed for the alkylated Oct4 peptide compared to its unmodified form . Derivatization of phosphopeptides by S , S ′‐dimethylthiobutanoylhydroxysuccinimide ester iodide provides longer retention times due to the presence of the hydrophobic propyl chain within the tag . Compared to unlabeled forms, elution into the mass spectrometer during LC‐ESI‐MS experiment with a higher fraction of organic solvent may improve ESI efficiencies.…”

Quantification of peptides using N‐terminal isotope coding and C‐terminal derivatization for sensitive analysis by micro liquid chromatography‐tandem mass spectrometry

“…The sensitive analysis of such peptides in biological samples with liquid chromatography‐electrospray ionization mass spectrometry (LC‐ESI‐MS) or tandem mass spectrometry (MS/MS) is essential for investigating their roles in biological processes and for peptide mapping in proteomics studies . In recent years, a number of derivatization methods for improving the ionization and detection of peptides during LC‐ESI‐MS analysis have been reported and applied for biological analysis; for example bovine serum albumin, transferrin, biotoxins, phosphopeptides and glycans . Concurrently, relative quantification or quantification using isotopically labeled compounds for accurate analysis is also important for protein and peptide studies.…”

Near-UV photodissociation of phosphopeptide cation-radicals

“…Both electron capture dissociation [28][29][30][31][32][33] and electron transfer dissociation (ETD) [34][35][36][37] have been used extensively for phosphopeptide sequencing. In particular, ETD has been explored and combined with CID in a parallel [38][39][40][41] or tandem fashion [42]. Other electron-involved ion activation methods use fast electron transfer from cesium atoms [43] and metastable atoms [44,45].…”