Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA.
Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC, Canada.
Methods Mol Biol. 2021;2228:353-384. doi: 10.1007/978-1-0716-1024-4_25.
The use of stable isotope-labeled standards (SIS) is an analytically valid means of quantifying proteins in biological samples. The nature of the labeled standards and their point of insertion in a bottom-up proteomic workflow can vary, with quantification methods utilizing curves in analytically sound practices. A promising quantification strategy for low sample amounts is external standard addition (ExSTA). In ExSTA, multipoint calibration curves are generated in buffer using serially diluted natural (NAT) peptides and a fixed concentration of SIS peptides. Equal concentrations of SIS peptides are spiked into experimental sample digests, with all digests (control and experimental) subjected to solid-phase extraction prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Endogenous peptide concentrations are then determined using the regression equation of the standard curves. Given the benefits of ExSTA in large-scale analysis, a detailed protocol is provided herein for quantifying a multiplexed panel of 125 high-to-moderate abundance proteins in undepleted and non-enriched human plasma samples. The procedural details and recommendations for successfully executing all phases of this quantification approach are described. As the proteins have been putatively correlated with various noncommunicable diseases, quantifying these by ExSTA in large-scale studies should help rapidly and precisely assess their true biomarker efficacy.
稳定同位素标记标准品（SIS）的使用是定量生物样品中蛋白质的一种分析有效手段。标记标准品的性质及其在自上而下的蛋白质组学工作流程中的插入点可能会有所不同，定量方法在分析上合理的实践中利用曲线。对于低样品量，一种有前途的定量策略是外部标准添加（ExSTA）。在 ExSTA 中，使用连续稀释的天然（NAT）肽和固定浓度的 SIS 肽在缓冲液中生成多点校准曲线。将等量的 SIS 肽掺入实验样品消化物中，所有消化物（对照和实验）在进行液相色谱串联质谱（LC-MS/MS）分析之前都要进行固相萃取。然后使用标准曲线的回归方程来确定内源性肽的浓度。鉴于 ExSTA 在大规模分析中的优势，本文提供了一种详细的方案，用于定量分析未耗尽和非富集的人类血浆样品中 125 种高到中等丰度的蛋白质的多重面板。描述了成功执行这种定量方法的所有阶段的详细过程和建议。由于这些蛋白质已被推测与各种非传染性疾病有关，因此通过 ExSTA 对这些蛋白质进行大规模研究应该有助于快速、准确地评估它们真正的生物标志物功效。
