磷酸肽和糖肽的库仑质谱绝对定量

IF 4.6 Q1 CHEMISTRY, ANALYTICAL
Md Tanim-Al Hassan, Yongling Ai, Bhavya Deshaboina, Timothy Yaroshuk, Arjun Sharma, Quentin Young, Howard D. Dewald and Hao Chen*, 
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引用次数: 0

摘要

磷酸化和糖基化是两种重要的蛋白质过渡后修饰(PTMs)。然而,由于缺乏蛋白质或肽标准,这些ptm的定量是具有挑战性的。在这项研究中,我们介绍了一种新的方法,使用库仑质谱法(CMS)来绝对定量磷酸肽和糖肽,而不使用标准品。首先,磷酸化的酪氨酸肽如TSTEPQpYQPGENL和RRLIEDAEpYAARG可以在CMS定量之前通过碱性磷酸酶酶解磷酸转化为电化学活性的酪氨酸肽。定量准确,定量误差小(0.3 ~ 6.6%)。另外,对于电化学无活性的磷酸肽和糖肽,用NHS酯试剂2,5-二氧基-1-吡啶基3,4-二羟基苯丙酸酯(DPDP)衍生化它们的n端,引入一个电活性儿茶酚标签,使DPDP衍生化的肽可以通过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,定量成功,定量误差小于6%。更进一步,我们应用这种方法来量化由NIST单克隆抗体(mAb)的胰蛋白酶消化产生的糖肽。通过亲水性相互作用液相色谱柱分离,成功地衍生了5个N297糖肽,并在不使用标准品的情况下,用CMS进行了分离和定量。鉴于PTMs的生物学意义,本研究对携带PTMs的多肽的定量研究将在定量蛋白质组学和生物学研究中具有很大的潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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.

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来源期刊
ACS Measurement Science Au
ACS Measurement Science Au 化学计量学-
CiteScore
5.20
自引率
0.00%
发文量
0
期刊介绍: ACS Measurement Science Au is an open access journal that publishes experimental computational or theoretical research in all areas of chemical measurement science. Short letters comprehensive articles reviews and perspectives are welcome on topics that report on any phase of analytical operations including sampling measurement and data analysis. This includes:Chemical Reactions and SelectivityChemometrics and Data ProcessingElectrochemistryElemental and Molecular CharacterizationImagingInstrumentationMass SpectrometryMicroscale and Nanoscale systemsOmics (Genomics Proteomics Metabonomics Metabolomics and Bioinformatics)Sensors and Sensing (Biosensors Chemical Sensors Gas Sensors Intracellular Sensors Single-Molecule Sensors Cell Chips Arrays Microfluidic Devices)SeparationsSpectroscopySurface analysisPapers dealing with established methods need to offer a significantly improved original application of the method.
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