Replacing immunoassays with tryptic digestion-peptide immunoaffinity enrichment and LC-MS/MS.
Replacing immunoassays with tryptic digestion-peptide immunoaffinity enrichment and LC-MS/MS.
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DOI：
10.4155/bio.11.319
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发表时间：
2012-02
期刊：
影响因子：
1.8
通讯作者：
Hoofnagle AN
中科院分区：
文献类型：
--
作者：
Becker JO;Hoofnagle AN
几十年来，免疫分析为临床医学和药物开发的治疗监测中的蛋白质生物标志物研究提供了框架。与此同时，研究人员发现了许多导致许多人类血清和血浆样本中的免疫测定不可靠的问题。蛋白质胰蛋白酶消化后的 LC-MS/MS 可能是一种有吸引力的解决方案，但该方法的灵敏度不足以直接测量许多重要的低丰度蛋白质。使用抗肽抗体对感兴趣的肽进行免疫富集可以提高该方法的灵敏度，大大简化基质，从而缩短色谱运行时间，并促进分析物的多重定量，从而降低复杂样本中定量蛋白质测量的成本。我们概述了开发检测所需的方法和步骤。此外，我们回顾了为使该方法更普遍适用而做出的努力。
For decades, immunoassays have provided the framework for protein biomarker studies in clinical medicine and in therapeutic monitoring for drug development. At the same time, investigators have uncovered many issues that make immunoassays unreliable in many human serum and plasma samples. LC-MS/MS after tryptic digestion of proteins is potentially an attractive solution, but the sensitivity of the method is not sufficient to measure many important low-abundance proteins directly. The use of antipeptide antibodies to immunoenrich peptides of interest can improve the sensitivity of the approach, greatly simplify the matrix enabling shortened chromatographic runs, and facilitate the multiplexed quantification of analytes, which could reduce the costs of quantitative protein measurements in complex specimens. We provide an overview of the method and the steps needed to develop an assay. In addition, we review the efforts to make this method generally more applicable.
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