Commercially available antibodies can be applied in quantitative multiplexed peptide immunoaffinity enrichment targeted mass spectrometry assays.
Commercially available antibodies can be applied in quantitative multiplexed peptide immunoaffinity enrichment targeted mass spectrometry assays.
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DOI：
10.1002/pmic.201500540
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发表时间：
2016-08
期刊：
影响因子：
3.4
通讯作者：
Paulovich, Amanda G.
中科院分区：
文献类型：
--
作者：
Schoenherr, Regine M.;Zhao, Lei;Ivey, Richard G.;Voytovich, Uliana J.;Kennedy, Jacob;Yan, Ping;Lin, Chenwei;Whiteaker, Jeffrey R.;Paulovich, Amanda G.
关键词：
肽的免疫亲和富集与多反应监测-质谱法（immuno-MRM）偶联，使得肽和翻译后修饰的高度特异性、灵敏度和精确定量成为可能。开发大量免疫-MRM测定的主要障碍是经验证用于肽的免疫亲和富集的单克隆抗体（mAb）的可用性差以及从头开发抗体的成本和前置时间。尽管商业上提供了数千种mAb，但很少有mAb被测试用于肽的免疫亲和富集。在这项研究中，我们测试了使用市售mAb进行肽免疫MRM测定的成功率。我们选择了105个商业mAb（76个靶向未修饰的“泛”表位，29个靶向磷酸化）与DNA损伤反应网络相关的蛋白质。我们发现，76个pan中的8个（11%）和29个磷酸特异性mAb中的5个（17%）从人细胞裂解物中捕获其蛋白质靶标的胰蛋白酶肽（通过LC-MS/MS检测）。这些mAb中的7种成功用于配置和分析表征免疫MRM测定。通过预先应用选择标准，结果表明，筛选成功率高达24%是可能的，建立了筛选大量目录抗体以提供易于获得的测定试剂的可行性。
Immunoaffinity enrichment of peptides coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM) enables highly specific, sensitive, and precise quantification of peptides and post-translational modifications. Major obstacles to developing a large number of immuno-MRM assays are the poor availability of monoclonal antibodies (mAbs) validated for immunoaffinity enrichment of peptides and the cost and lead time of developing the antibodies de novo. Although many thousands of mAbs are commercially offered, few have been tested for application to immunoaffinity enrichment of peptides. In this study we tested the success rate of using commercially available mAbs for peptide immuno-MRM assays. We selected 105 commercial mAbs (76 targeting non-modified “pan” epitopes, 29 targeting phosphorylation) to proteins associated with the DNA damage response network. We found that 8 of the 76 pan (11%) and 5 of the 29 phospho-specific mAbs (17%) captured tryptic peptides (detected by LC-MS/MS) of their protein targets from human cell lysates. Seven of these mAbs were successfully used to configure and analytically characterize immuno-MRM assays. By applying selection criteria upfront, the results indicate that a screening success rate of up to 24% is possible, establishing the feasibility of screening a large number of catalog antibodies to provide readily-available assay reagents.
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影响因子：
7
作者：
Whiteaker, Jeffrey R.;Zhao, Lei;Paulovich, Amanda G.
通讯作者：
Paulovich, Amanda G.
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Schoenherr, Regine M.;Saul, Richard G.;Paulovich, Amanda G.
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Kennedy, Jacob J.;Abbatiello, Susan E.;Paulovich, Amanda G.
通讯作者：
Paulovich, Amanda G.