Use of Stable Isotope Dimethyl Labeling Coupled to Selected Reaction Monitoring to Enhance Throughput by Multiplexing Relative Quantitation of Targeted Proteins

In this manuscript, we present a proof-of-concept study for targeted relative protein quantitation workflow using chemical labeling in the form of dimethylation, coupled with selected reaction monitoring (dimethyl-SRM). We first demonstrate close to complete isotope incorporation for all peptides tested. The accuracy, reproducibility, and linear dynamic range of quantitation are further assessed based on known ratios of nonhuman standard proteins spiked into human cerebrospinal fluid (CSF) as a model complex matrix. Quantitation reproducibility below 20% (CV < 20%) was obtained for analyte concentrations present at a dynamic range of 4 orders of magnitude lower than that of the background proteins. An error of less than 15% was observed when measuring the abundance of 44 out of 45 major human plasma proteins. Dimethyl-SRM was further examined by comparing the relative quantitation of eight proteins in human CSF with the relative quantitation obtained using synthetic heavy peptides coupled to stable isotope dilution-SRM (SID-SRM). Comparison between the two methods reveals that the correlation between dimethyl-SRM and SID-SRM is within 0.3–33% variation, demonstrating the accuracy of relative quantitation using dimethyl-SRM. Dimethyl labeling coupled with SRM provides a fast, convenient, and cost-effective alternative for relative quantitation of a large number of candidate proteins/peptides.本研究针对采用二甲基化化学标记结合选择反应监测（Selected Reaction Monitoring，SRM）的靶向相对蛋白质定量工作流程（dimethyl-SRM）开展了一项概念验证研究（proof-of-concept study）。本研究首先证实，所有待测肽段均实现了接近完全的同位素掺入。本研究进一步以掺入非人源标准蛋白的人脑脊液（cerebrospinal fluid，CSF）作为复杂基质模型，基于已知的蛋白掺入比例，对定量的准确性、重现性及线性动态范围进行了评估。对于浓度比背景蛋白低4个数量级的分析物，其定量重现性可控制在20%以内（变异系数（Coefficient of Variation，CV）< 20%）。在检测45种主要人血浆蛋白中的44种的丰度时，检测误差均低于15%。本研究进一步通过将人脑脊液中8种蛋白的相对定量结果，与采用合成重质肽段结合稳定同位素稀释-SRM（stable isotope dilution-SRM，SID-SRM）得到的定量结果进行对比，对dimethyl-SRM方法进行了验证。两种方法的对比结果显示，dimethyl-SRM与SID-SRM的相关性偏差处于0.3%~33%区间内，证实了dimethyl-SRM用于相对定量的准确性。二甲基化标记结合SRM技术，可为大量候选蛋白/肽段的相对定量提供一种快速、便捷且经济高效的替代方案。