Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum
Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum
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DOI：
10.1016/j.clinbiochem.2012.12.019
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发表时间：
2013-04-01
影响因子：
2.8
通讯作者：
Lopez, Mary F.
中科院分区：
文献类型：
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作者：
Krastins, Bryan;Prakash, Amol;Lopez, Mary F.
目的：本研究的目的是开发针对人血浆或血清中临床重要蛋白质的高通量、定量和高选择性质谱、靶向免疫测定。设计和方法：所描述的方法将质谱免疫测定（MSIA）（一种先前开发的在用抗蛋白质抗体激活并固定在移液管吸头中的整体微柱上进行免疫富集的技术）与选择性反应监测（SRM）检测和目标肽（包括临床相关肽）的精确定量相结合。结果：在本报告中，我们展示了针对 16 种不同靶蛋白的 MSIA-SRM 检测的快速发展，涵盖七个不同的临床重要领域（包括神经、阿尔茨海默病、心血管、内分泌功能、癌症和其他疾病），浓度范围从 pg/mL 到 mg/mL。所报道的 MSIA-SRM 测定法表现出高灵敏度（在已发表的临床范围内）、精确性、稳健性和高通量，以及对许多目标蛋白的临床相关亚型的特异性检测。大多数测定均使用真实的临床样本进行了测试。此外，MSIA-SRM 测定数据与临床样本队列中甲状旁腺激素 (PTH) 和胰岛素生长因子 1 (IGF1) 的临床分析仪测量值之间存在正相关性（R2 0.67-0.87，具体取决于目标肽）。结论：我们提出了一种实用且可扩展的方法，用于快速开发和部署基于 MS 的 SRM 测定，用于临床相关蛋白质和测量的水平真实临床样品中的目标分析物。该方法允许对单个蛋白质亚型进行特异性定量，并解决临床蛋白质组学应用中蛋白质异质性的难题。 (C) 2013 年加拿大临床化学家协会。由爱思唯尔公司出版。保留所有权利。
Objectives: The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum.Design and methods: The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants.Results: In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples.In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts.Conclusions: We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications. (C) 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.