Percy Andrew J, Yang Juncong, Chambers Andrew G, Mohammed Yassene, Miliotis Tasso, Borchers Christoph H
University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC, V8Z 7X8, Canada.
Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333 ZA, Leiden, Netherlands.
Adv Exp Med Biol. 2016;919:515-530. doi: 10.1007/978-3-319-41448-5_24.
Quantitative mass spectrometry (MS)-based approaches are emerging as a core technology for addressing health-related queries in systems biology and in the biomedical and clinical fields. In several 'omics disciplines (proteomics included), an approach centered on selected or multiple reaction monitoring (SRM or MRM)-MS with stable isotope-labeled standards (SIS), at the protein or peptide level, has emerged as the most precise technique for quantifying and screening putative analytes in biological samples. To enable the widespread use of MRM-based protein quantitation for disease biomarker assessment studies and its ultimate acceptance for clinical analysis, the technique must be standardized to facilitate precise and accurate protein quantitation. To that end, we have developed a number of kits for assessing method/platform performance, as well as for screening proposed candidate protein biomarkers in various human biofluids. Collectively, these kits utilize a bottom-up LC-MS methodology with SIS peptides as internal standards and quantify proteins using regression analysis of standard curves. This chapter details the methodology used to quantify 192 plasma proteins of high-to-moderate abundance (covers a 6 order of magnitude range from 31 mg/mL for albumin to 18 ng/mL for peroxidredoxin-2), and a 21-protein subset thereof. We also describe the application of this method to patient samples for biomarker discovery and verification studies. Additionally, we introduce our recently developed Qualis-SIS software, which is used to expedite the analysis and assessment of protein quantitation data in control and patient samples.
基于定量质谱（MS）的方法正在成为系统生物学以及生物医学和临床领域中解决与健康相关问题的核心技术。在几个“组学”学科（包括蛋白质组学）中，一种以蛋白质或肽水平的稳定同位素标记标准品（SIS）为基础的选择反应监测（SRM）或多反应监测（MRM）-MS为中心的方法，已成为定量和筛选生物样品中假定分析物的最精确技术。为了使基于MRM的蛋白质定量能够广泛应用于疾病生物标志物评估研究，并最终被临床分析所接受，该技术必须标准化，以促进精确和准确的蛋白质定量。为此，我们开发了许多试剂盒，用于评估方法/平台性能，以及筛选各种人类生物流体中提出的候选蛋白质生物标志物。总体而言，这些试剂盒采用自下而上的液相色谱-质谱方法，以SIS肽作为内标，并使用标准曲线的回归分析对蛋白质进行定量。本章详细介绍了用于定量192种高至中等丰度血浆蛋白（涵盖从白蛋白的31mg/mL到过氧化物还原酶-2的18ng/mL的6个数量级范围）及其21种蛋白质子集的方法。我们还描述了该方法在患者样本中的应用，用于生物标志物发现和验证研究。此外，我们介绍了我们最近开发的Qualis-SIS软件，该软件用于加快对照和患者样本中蛋白质定量数据的分析和评估。
