High-throughput proteomics using submicroliter amounts of plasma for comprehensive assessment of the immune status

总结 通常，进行许多不同的测试以获得先天性和先天性疾病的多参数评估。 适应性免疫状态这类检查通常包括红细胞沉降率、C反应蛋白 (CRP)、降钙素原、各种补体途径蛋白、总抗体（Ab）同种型和/或 亚类和特定Ab滴度。所有这些测试都是在不同的分析平台上进行的 每次测试需要单独的等分试样。因此，即使每次测试的样本消耗量不大， 是多次独立测试所需的大量累积体积。虽然这些样品 对于健康的青少年和成年人来说，这些要求可能并不令人望而却步， 在特别脆弱的人群中具有挑战性，如早产儿和体弱和/或老年人 个体对于这些人群，广泛的不同测试通常非常重要，然而， 样本往往是有限的。 与资助机会的目的一致，“开发用于 监测免疫反应”，我们建议将四种不同的蛋白质检测转移到一个共同的 处理和分析平台，其可以在相同的亚微升血浆等分试样上进行 从而显著地减少了样品体积需求。四种感兴趣的蛋白质测定 包括i）补体成分扩展组，ii）CRP，iii）抗体同种型和亚类 包括一个新的和创新的免疫球蛋白结构域解析的免疫球蛋白相关蛋白质的地图 库（又称IgOme），和iv）经典血浆蛋白质组，即约400种充分描述的蛋白质， 其中免疫功能明显。来自所有这些测定的定量信息将提供 我们的免疫状态的精美地图，一个假设，我们将通过相关的测试，我们的 B型肝炎疫苗接种前后抗B型肝炎抗体滴度>500 新生儿样本将由波士顿儿童医院的NIAID资助的HIPC（人类 免疫学项目联盟），这将大大增加这一队列的影响，超越其 原始范围。考虑到这次资助机会对样品保留的要求，我们 将推进我们的血浆蛋白质组学管道，使吞吐量增加四倍，达到每天至少96个样本 并将血浆体积要求四分之一至<100纳升。 因此，在未来，1微升血浆将足以进行数十次时间独立的LC/MS。 基于蛋白质分析，导致数百种蛋白质的定量信息， 来自最小和/或最虚弱患者的微量血浆将足以建立 一个精确详细和公正的分子图的免疫状态的任何人。

SUMMARY Often, many different tests are performed to obtain a multiparameter assessment of the innate and adaptive immune status. Such tests often include erythrocyte sedimentation rate, C-reactive protein (CRP), procalcitonin, various complement pathway proteins, total antibody (Ab) isotypes and/or subclasses, and specific Ab titers. All these tests are performed on different analytical platforms requiring a separate aliquot for each test. Thus, even with modest sample consumption per test, there is a substantial cumulative volume required for multiple independent tests. While these sample requirements may not be prohibitive for healthy adolescents and adults, they are particularly challenging in especially vulnerable populations such as premature newborns, and frail and/or elderly individuals. For these populations, a wide range of different tests is often very important, however, samples are often limited. In agreement with the purpose of the funding opportunity “Development of Sample Sparing Assays for Monitoring Immune Responses”, we propose to move four different protein assays onto a common processing and analysis platform, which can be performed on the same submicroliter aliquot of plasma thereby significantly reducing the sample volume requirements. The four protein assays of interest include i) an expanded panel of complement components, ii) CRP, iii) antibody isotypes and subclasses including a novel and innovative immunoglobulin domain-resolved map of the IgOme-related protein repertoire (aka IgOme), and iv) the classical plasma proteome, i.e. ~400 well described proteins many of which of clear immunological function. The quantitative information from all these assays will provide us with an exquisite map of the immune status, a hypothesis that we will be tested by correlating our findings with the anti-hepatitis B titer measured before and after hepatitis B vaccination in >500 newborns. Samples will be provided by Boston Children's Hospital's NIAID-funded HIPC (Human Immunology Project Consortium), which will significantly increase the impact of this cohort beyond its original scope. Taking the request of this funding opportunity for sample sparing to a different level, we will advance our plasma proteomics pipeline to quadruple the throughput to at least 96 samples/day and to quarter the plasma volume requirements to <100 nanoliter. Thus, in the future, 1 microliter of plasma will be sufficient for tens of temporally independent LC/MS- based protein assays, resulting in quantitative information for hundreds of proteins such that even a miniscule amount of plasma from the smallest and/or most frail patients will be sufficient to establish an exquisitely detailed and unbiased molecular map of immune status of any person.