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

摘要 通常，执行许多不同的测试以获得对先天和 适应性免疫状态。这类测试通常包括血沉、C反应蛋白 (CRP)、降钙素原、各种补体途径蛋白、总抗体(Ab)亚型和/或 亚类和特异性抗体效价。所有这些测试都在不同的分析平台上进行 每一次测试都需要单独的等分。因此，即使每次测试消耗的样本不多，也有 是多个独立测试所需的大量累积容量。而这些样品 对于健康的青少年和成年人来说，要求可能并不是令人望而却步的，它们特别是 在特别脆弱的人群中具有挑战性，如早产儿、体弱者和/或老年人 个人。然而，对于这些人群来说，一系列不同的测试往往非常重要， 样本通常是有限的。 与资助机会的目的一致“为以下目的制定样本保留分析 监测免疫反应“，我们建议将四种不同的蛋白质分析移到共同的 处理和分析平台，可以在相同的亚微升等量血浆上执行 从而大大降低了样品量要求。感兴趣的四种蛋白质分析方法 包括一)扩大补体组分，二)C反应蛋白，三)抗体亚型和亚类 包括一种新的创新的IgOme相关蛋白的免疫球蛋白结构域解析图 曲目(又名IgOme)，和iv)经典的血浆蛋白质组，即~400多个描述良好的蛋白质 其中有明确的免疫功能。所有这些化验的定量信息将提供 我们有一张精美的免疫状态图，这是一个假设，我们将通过将我们的 500人乙肝疫苗接种前后抗-乙肝效价检测结果分析 新生儿。样本将由波士顿儿童医院NIAID资助的HIPC(Human 免疫学项目联盟)，这将显著增加这一队列的影响，超出其 原始作用域。将这次资助机会的要求提升到一个不同的水平，我们 将推进我们的血浆蛋白质组流水线，使产量翻两番，达到每天至少96个样品 并将等离子体体积需求的四分之一提高到100纳升。 因此，在未来，1微升的血浆将足以用于数十个时间上独立的LC/MS- 基于蛋白质分析，导致数百种蛋白质的定量信息，即使是一个 从最小和/或最虚弱的患者身上采集微量血浆就足以确定 任何人的免疫状态的精致详细和公正的分子图谱。

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.