Department of Chemistry & Environmental Science, New Jersey Institute of Technology, Newark, New Jersey 07102, United States.
Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio 45701, United States.
Anal Chem. 2020 Jun 2;92(11):7877-7883. doi: 10.1021/acs.analchem.0c01151. Epub 2020 May 15.
Accurate quantification is essential in the fields of proteomics, clinical assay, and biomarker discovery. Popular methods for absolute protein quantitation by mass spectrometry (MS) involve the digestion of target protein and employ isotope-labeled peptide internal standards to quantify chosen surrogate peptides. Although these methods have gained success, syntheses of isotope-labeled peptides are time-consuming and costly. To eliminate the need for using standards or calibration curves, herein we present a coulometric mass spectrometric (CMS) approach for absolute protein quantitation, based on the electrochemical oxidation of a surrogate peptide combined with mass spectrometric measurement of the oxidation yield. To demonstrate the utility of this method, several proteins were analyzed such as model proteins β-casein, and apomyoglobin as well as circadian clock protein KaiB isolated from . In our experiment, tyrosine-containing peptides were selected as surrogate peptides for quantitation, considering the oxidizable nature of tyrosine. Our data showed that the results for surrogate peptide quantity measured by our method and by traditional isotope dilution method are in excellent agreement, with the discrepancy of 0.3-3%, validating our CMS method for absolute quantitation. Furthermore, therapeutic monoclonal antibody (mAb) could be quantified by our method as well. Due to the high specificity and sensitivity of MS and no need to use isotope-labeled peptide standards, our CMS method would be of high value for the absolute proteomic quantification.
准确的定量分析在蛋白质组学、临床检测和生物标志物发现等领域至关重要。目前，基于质谱（MS）的绝对蛋白质定量的常用方法涉及目标蛋白质的消化，并采用同位素标记肽内标来定量选择的替代肽。尽管这些方法已经取得了成功，但同位素标记肽的合成既耗时又昂贵。为了消除对使用标准品或校准曲线的需求，本研究提出了一种基于电化学氧化替代肽并结合质谱测量氧化产率的库仑质谱（CMS）绝对蛋白质定量方法。为了验证该方法的实用性，我们分析了几种蛋白质，如模型蛋白 β-酪蛋白和脱辅基肌红蛋白，以及从 中分离出的生物钟蛋白 KaiB。在我们的实验中，选择含有酪氨酸的肽作为定量替代肽，考虑到酪氨酸的可氧化性质。我们的数据表明，我们的方法和传统同位素稀释法测量替代肽数量的结果非常吻合，差异在 0.3-3%之间，验证了我们的 CMS 方法用于绝对定量的有效性。此外，我们的方法也可以定量治疗性单克隆抗体（mAb）。由于 MS 具有高特异性和灵敏度，并且不需要使用同位素标记肽标准品，因此我们的 CMS 方法对于绝对蛋白质组定量具有很高的价值。
