Tanim-Al Hassan Md, Ai Yongling, Deshaboina Bhavya, Yaroshuk Timothy, Sharma Arjun, Young Quentin, Dewald Howard D, Chen Hao
Department of Chemistry and Environmental Science, New Jersey Institutes of Technology, University Heights, Newark, New Jersey 07102, United States.
Chemistry & Biochemistry Department, Ohio University, Athens, Ohio 45701, United States.
ACS Meas Sci Au. 2025 Jul 18;5(4):559-571. doi: 10.1021/acsmeasuresciau.5c00047. eCollection 2025 Aug 20.
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.
磷酸化和糖基化是两种重要的蛋白质翻译后修饰（PTM）。然而，由于缺乏蛋白质或肽标准品，对这些PTM进行定量具有挑战性。在本研究中，我们引入了一种使用库仑质谱（CMS）的新方法，用于在不使用标准品的情况下对磷酸肽和糖肽进行绝对定量。首先，在CMS定量之前，使用碱性磷酸酶通过酶促脱磷酸作用，将磷酸化的酪氨酸肽（如TSTEPQpYQPGENL和RRLIEDAEpYAARG）转化为具有电化学活性的酪氨酸肽。以较小的定量误差（0.3 - 6.6%）获得了准确的定量结果。或者，对于无电化学活性的磷酸肽和糖肽，用NHS酯试剂2,5 - 二氧代 - 1 - 吡咯烷基3,4 - 二羟基苯丙酸酯（DPDP）对其N端进行衍生化，以引入一个电活性儿茶酚标签，使DPDP衍生化的肽能够通过CMS进行定量。该策略首先使用肽RGD、GGYR、磷酸肽RRApSVA和糖肽NYIVGQPSS - (β - GlcNAc) - TGNL - OH进行验证，实现了小于6%的定量误差的成功定量。更进一步，我们应用此方法对美国国家标准与技术研究院单克隆抗体（mAb）胰蛋白酶消化产生的糖肽进行定量。通过亲水相互作用液相色谱柱分离，成功地对五个N297糖肽进行了衍生化、分离和CMS定量，且无需使用标准品。由于PTM的生物学意义，本研究中对携带PTM的肽进行定量在定量蛋白质组学和生物学研究方面具有很高的潜力。
