Quantitative Mass Spectrometric Immunoassays for Population Proteomics

描述（由申请人提供）：该I期项目的主要目标是开发β -2微球蛋白、胱抑素C、转铁蛋白、转甲状腺素和视黄醇结合蛋白的定量质谱免疫分析。这五种蛋白质最近在1000个健康人群中通过定性质谱免疫分析进行了检测，检测到（没有提供浓度信息）许多蛋白质变异。由于基于三明治的免疫测定（如elisa）不能特异性地定量细微的蛋白质修饰，在本项目中，我们建议开发定量质谱免疫测定这些蛋白质及其同种异构体。将仔细选择内部参比标准品，并逐步进行标准曲线定量质谱免疫分析开发。定量质谱免疫测定的精度（测定内和测定间）、线性、灵敏度和特异性将被确定。作为最后的验证步骤，定量MSIA分析将与现有的ELISA分析进行评估和基准测试。本文提出的定量质谱免疫测定将提供独特和直接的方法来定量蛋白质变异，当应用于疾病队列时，可以提供有价值的见解，了解这些变异在疾病的发生、进展和治疗反应中的作用。公共卫生相关性：β -2-微球蛋白、胱抑制素C、转铁蛋白、转甲状腺素和视黄醇结合蛋白的定量质谱免疫测定的发展将使这些蛋白及其修饰模式的变化可能与疾病相关的常规定量监测成为可能。有了新的检测方法，我们将更好地研究这些蛋白质修饰在病理过程中的作用，并评估它们作为疾病发生和进展以及对治疗反应的新指标的潜力。

DESCRIPTION (provided by applicant): The main objective of this Phase I project is to develop quantitative Mass Spectrometric Immunoassays for beta-2-microglobulin, cystatin C, transferrin, transthyretin, and retinol binding protein. These five proteins were recently assayed via qualitative mass spectrometric immunoassays in 1,000 healthy cohorts, detecting (without providing concentration information) numerous protein variant. Because sandwich-based immunoassays such as ELISAs cannot specifically quantify the subtle protein modifications, in this project we propose to develop quantitative mass spectrometric immunoassays for these proteins and their isoforms. An internal reference standard will be carefully chosen, and a step-by-step standard curve quantitative mass spectrometric immunoassay development will be undertaken. The precision (intra-assay and inter-assay), linearity, sensitivity, and specificity of the quantitative mass spectrometric immunoassays will be determined. As a final validation step, the quantitative MSIA assays will be evaluated and benchmarked against existing ELISA assays. The quantitative mass spectrometric immunoassays proposed here will offer unique and straightforward means of quantifying protein variants, and when applied to disease cohorts, can provide valuable insight into the role of these variants in the onset of the disease, progression, and response to therapy. PUBLIC HEALTH RELEVANCE: The development of quantitative Mass Spectrometric Immunoassays for beta-2- microglobulin, cystatin C, transferrin, transthyretin, and retinol binding protein will enable routine quantitative monitoring of these proteins and the changes in their modifications patterns that might be relevant to disease. With the new assays in hand, we will be better equipped to study the effects of these proteins' modifications in pathological processes and evaluate their potential as new indicators of the onset and progression of diseases, and response to therapy.