Dynein axonemal intermediate chain 1 protein (DNAI1) plays an essential role in cilia structure and function, while its mutations lead to primary ciliary dyskinesia (PCD). Accurate quantitation of DNAI1 in lung tissue is crucial for comprehensive understanding of its involvement in PCD, as well as for developing the potential PCD therapies. However, the current protein quantitation method is not sensitive enough to detect the endogenous level of DNAI1 in complex biological matrix such as lung tissue. In this study, a quantitative method combining immunoprecipitation with nanoLC-MS/MS was developed to measure the expression level of human wild-type (WT) DNAI1 protein in lung tissue. To our understanding, it is the first immunoprecipitation (IP)-MS based method for absolute quantitation of DNAI1 protein in lung tissue. The DNAI1 quantitation was achieved through constructing a standard curve with recombinant human WT DNAI1 protein spiked into lung tissue matrix. This method was qualified with high sensitivity and accuracy. The lower limit of quantitation of human DNAI1 was 4 pg/mg tissue. This assay was successfully applied to determine the endogenous level of WT DNAI1 in human lung tissue. The results clearly demonstrate that the developed assay can accurately quantitate low-abundance WT DNAI1 protein in human lung tissue with high sensitivity, indicating its high potential use in the drug development for DNAI1 mutation-caused PCD therapy.

中文翻译：

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使用基于 nanoLC-PRM-MS 的靶向蛋白质组学方法结合免疫沉淀对肺组织中的人野生型 DNAI1 蛋白进行绝对定量

动力蛋白轴丝中间链 1 蛋白 (DNAI1) 在纤毛结构和功能中发挥着重要作用，而其突变会导致原发性纤毛运动障碍 (PCD)。肺组织中 DNAI1 的准确定量对于全面了解其与 PCD 的关系以及开发潜在的 PCD 疗法至关重要。然而，目前的蛋白质定量方法不够灵敏，无法检测肺组织等复杂生物基质中DNAI1的内源水平。在本研究中，开发了一种将免疫沉淀与nanoLC-MS/MS相结合的定量方法来测量肺组织中人野生型（WT）DNAI1蛋白的表达水平。据我们了解，这是第一个基于免疫沉淀 (IP)-MS 的肺组织中 DNAI1 蛋白绝对定量方法。 DNAI1 定量是通过将重组人 WT DNAI1 蛋白掺入肺组织基质中构建标准曲线来实现的。该方法具有较高的灵敏度和准确度。人 DNAI1 的定量下限为 4 pg/mg 组织。该测定已成功应用于测定人肺组织中 WT DNAI1 的内源水平。结果清楚地表明，所开发的检测方法可以高灵敏度地准确定量人肺组织中的低丰度 WT DNAI1 蛋白，表明其在 DNAI1 突变引起的 PCD 治疗的药物开发中具有很高的潜在用途。