Research Centre for Toxic Compounds in the Environment , Masaryk University , Brno , Czech Republic.
Department of Clinical Hematology , University Hospital Brno , Brno , Czech Republic.
J Proteome Res. 2019 Jan 4;18(1):380-391. doi: 10.1021/acs.jproteome.8b00657. Epub 2018 Nov 20.
Inflammation is the first line defense mechanism against infection, tissue damage, or cancer development. However, inappropriate inflammatory response may also trigger diseases. The quantification of inflammatory proteins is essential to distinguish between harmful and beneficial immune response. Currently used immunoanalytical assays may suffer specificity issues due to antigen-antibody interaction and possible cross-reactivity of antibody with other protein species. In addition, immunoanalytical assays typically require invasive blood sampling and additional logistics; they are relatively costly and highly challenging to multiplex. We present a multiplex assay based on selected reaction monitoring (SRM) for quantification of seven acute-phase proteins (i.e., SAA1, SAA2-isoform1, SAA4, CRP, A1AT-isoform1, A1AG1, A1AG2) and the adaptive immunity effector IGHA1 in dried blood spots. This type of sample is readily available from all human subjects including newborns. The study utilizes proteotypic isotopically labeled peptides with trypsin-cleavable tag and presents optimized and reproducible workflow and several important practical remarks regarding quantitative SRM assays development. The panel of inflammatory proteins was quantified with sequence specificity capable to differentiate protein isoforms with intra- and interday precision (<16.4% coefficient of variation (CV) and <14.3% CV, respectively). Quantitative results were correlated with immuno-nephelometric assay (typically greater than 0.9 Pearson's R).
炎症反应是对抗感染、组织损伤或癌症发展的第一道防线机制。然而，不适当的炎症反应也可能引发疾病。炎症蛋白的定量对于区分有害和有益的免疫反应至关重要。目前使用的免疫分析测定可能会因抗原-抗体相互作用和抗体与其他蛋白质物种的可能交叉反应而存在特异性问题。此外，免疫分析测定通常需要侵入性的血液采样和额外的物流；它们相对昂贵，并且高度难以实现多重检测。我们提出了一种基于选择反应监测（SRM）的多重测定法，用于定量检测七种急性期蛋白（即 SAA1、SAA2-isoform1、SAA4、CRP、A1AT-isoform1、A1AG1、A1AG2）和适应性免疫效应物 IGHA1 在干血斑中的含量。这种类型的样本很容易从所有人类受试者中获得，包括新生儿。该研究利用具有胰蛋白酶可切割标签的蛋白质特征同位素标记肽，并提出了优化和可重复的工作流程，以及关于定量 SRM 测定开发的几个重要实际注意事项。该炎症蛋白组学的定量分析具有序列特异性，能够区分具有日内和日间精密度（<16.4%变异系数（CV）和<14.3%CV）的蛋白质同工型。定量结果与免疫比浊测定具有相关性（通常大于 0.9 Pearson's R）。
