Absolute Quantitation of Phosphopeptides and Glycopeptides Using Coulometric Mass Spectrometry

Phosphorylation and glycosylation are two important protein post-transitional modifications (PTMs). However, quantification of these PTMs is challenging due to the lack of protein or peptide standards. In this study, we introduced a novel approach using coulometric mass spectrometry (CMS) for absolute quantitation of phosphopeptides and glycopeptides without using standards. First, phosphorylated tyrosine peptides such as TSTEPQpYQPGENL and RRLIEDAEpYAARG can be converted into electrochemically active tyrosine peptides via enzymatic phosphate removal using alkaline phosphatase prior to CMS quantitation. Accurate quantitation was obtained with small quantitation errors (0.3-6.6%). Alternatively, for electrochemically inactive phosphopeptides and glycopeptides, derivatization of their N-termini with an NHS ester reagent, 2,5-dioxo-1-pyrrolidinyl 3,4-dihydroxybenzene propanoate (DPDP), was conducted to introduce one electroactive catechol tag, allowing the DPDP-derivatized peptides to be quantified by CMS. This strategy was first validated using peptides RGD, GGYR, phosphopeptide RRApSVA, and glycopeptide NYIVGQPSS-(β-GlcNAc)-TGNL-OH, and successful quantification was achieved with quantification errors less than 6%. Taking one step further, we applied this approach to quantify glycopeptides generated from tryptic digestion of the NIST monoclonal antibody (mAb). Through hydrophilic interaction liquid chromatography column separation, five N297 glycopeptides were successfully derivatized, separated, and quantified by CMS without the use of standards. Due to the biological significance of PTMs, this study for quantifying peptides carrying PTMs would have a high potential for quantitative proteomics and biological research.

Abstract licence: CC BY-NC-ND

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