Evaluation of Large Scale Quantitative Proteomic Assay Development Using Peptide Affinity-based Mass Spectrometry
Evaluation of Large Scale Quantitative Proteomic Assay Development Using Peptide Affinity-based Mass Spectrometry
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DOI：
10.1074/mcp.m110.005645
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发表时间：
2011-04-01
影响因子：
7
通讯作者：
Paulovich, Amanda G.
中科院分区：
文献类型：
--
作者：
Whiteaker, Jeffrey R.;Zhao, Lei;Paulovich, Amanda G.
稳定同位素标准品和抗肽抗体捕获 (SISCAPA) 将肽的亲和富集与稳定同位素稀释和多反应监测质谱检测相结合，以提供作为各自蛋白质替代物的肽的定量测量。在本报告中，我们描述了一项可行性研究，以确定生产适合 SISCAPA 测定的抗体的成功率，以便为大规模测定开发策略提供信息。设计了一个工作流程，其中包括多重免疫策略，其中使用来自单个蛋白质靶标的多达五种蛋白肽来免疫个体兔子。总共 403 种代表 89 个蛋白质靶标的蛋白型胰蛋白酶肽被用作免疫原。通过ELISA测量抗肽抗体滴度，并选择代表89种蛋白质的220种抗肽抗体进行亲和纯化。使用胰蛋白酶消化的人血浆基质对这些抗体在 SISCAPA 多重反应监测测定中的性能进行了表征。超过一半的检测能够检测人血浆中浓度低于 0.5 fmol/μl 的目标肽，对应于低于 100 ng/ml 的蛋白质浓度。在本次评估研究中，多重五肽免疫原的策略成功地生成了 100% 目标蛋白的工作检测方法。这些结果表明，单个实验室每年开发数百种检测方法是可行的，并可以规划具有成本效益的 SISCAPA 检测方法。分子与细胞蛋白质组学 10：10.1074/mcp.M110.005645，1-10，2011。
Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/mu l in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.005645, 1-10, 2011.