Immunoplate-MALDI MS platform and assays

项目总结 这项提议的目标是开发简单、健壮和经济高效的基于质谱学(MS)的 结合酶免疫分析最佳方面的蛋白质分析和平台--平板蛋白质 免疫亲和捕获，通过MALDI-TOF MS提供的直接MS检测，这些免疫板- MALDI MS分析是酶免疫分析的改进版本，其中辅助报告 抗体检测步骤被MS检测取代，从而实现了明确的蛋白质检测和 通过对其质量的具体和准确测量进行识别。 新平台和检测方法发展的动力是检测和研究人类的能力 蛋白质形式--单个基因的蛋白质产物存在的不同分子形式。质量 光谱分析是唯一能够提供单个蛋白质组分的测量方法，这些组分 原始的蛋白质序列。这些蛋白形式最好完整检测，这一点很容易通过MALDI- 亲和力回收的蛋白质的MALDI质谱图显示了由 抗体包被的免疫板效果良好。蛋白质形式的相对比例可以从它们的信号中确定 质谱图，并报告为丰度百分比。新的平台和检测将满足三个关键 MS蛋白质分析的促成因素：含量、成本和简单性。 在这项第一阶段的研究中，我们将通过原理证明免疫平板-MALDI MS分析来展示该平台 对于六种呈现蛋白质形式的蛋白质生物标志物：载脂蛋白E、转铁蛋白、转甲状腺蛋白、胱抑素C、视黄醇 结合蛋白和B型利钠肽。这些蛋白质生物标志物涵盖了广泛的 浓度和分子量，这将使我们能够测试平台范围、能力和 限制。将实现从人血浆样本中免疫亲和分离蛋白质 抗体包被的96孔板，之后捕获的蛋白质及其蛋白形式将被洗脱和 在用于MS分析的MALDI目标上被发现。将对实验条件进行抗体吸附测试， 人血浆分析和MALDI靶标的洗脱。步骤的数量将保持在最低水平，但 足以以尽可能低的成本获得令人满意的性能。新化验方法的重复性 将进行评估，并对化验结果进行基准测试。 我们的目标是开发面向一般研发市场的免疫盘-MALDI MS平台。化验设计于 这一平台将具有成本效益和简单性，可以很容易地被现有MALDI的实验室采用 MS仪器。新的平台和分析可以用于蛋白质形式的发现工作，以及 更大规模的临床验证研究，以描绘特定蛋白形式的临床相关性。

PROJECT SUMMARY The goal of this proposal is to develop simple, robust and cost-effective Mass Spectrometry (MS)-based protein assays and platform by combining the best aspects of enzymatic immunoassays – plate based protein immunoaffinity capture, with the straightforward MS detection offered by MALDI-TOF MS. These immunoplate- MALDI MS assays are improved version of the enzymatic immunoassays wherein the secondary reporter antibody detection step is replaced with MS detection, enabling unambiguous protein detection and identification through specific and accurate measurements of its mass. The impetus for the development of the new platform and assays is the ability to detect and study human proteoforms - the different molecular forms in which the protein product of a single gene exist. Mass spectrometry is uniquely capable of providing measurements of individual proteoforms masses that vary from the original protein sequence. These proteoforms are best detected intact, which is readily achieved by MALDI- TOF MS. A MALDI mass spectrum of an affinity–retrieved protein displays all proteoforms captured by an antibody-coated immunoplate well. Relative ratios of the proteoforms can be determined from their signals in the mass spectra, and reported as % abundance. The new platform and assays will meet the three key enabling factors for MS protein assays: content, cost, and simplicity. In this Phase I research we will demonstrate the platform via proof-of-principle immunoplate-MALDI MS assays for six protein biomarkers that exhibit proteoforms: apolipoprotein E, transferrin, transthyretin, cystatin C, retinol binding protein, and B-type natriuretic peptide. These protein biomarkers span a wide spectrum of concentrations and molecular weights, which will enable us to test the platform range, capabilities and limitations. Immunoaffinity isolation of the proteins from human plasma samples will be achieved utilizing antibody-coated 96-well plates, after which the captured proteins and their proteoforms will be eluted and spotted on a MALDI target for MS analyses. Experimental conditions will be tested for the antibody adsorption, human plasma assaying, and elution onto MALDI targets. The number of steps will be kept to a minimum, yet enough to obtain a satisfactory performance at the lowest cost possible. The reproducibility of the new assays will be evaluated, and the assays will be benchmarked. Our goal is to develop the immunoplate-MALDI MS platform for the general R&D market. Assay designed on this platform will be cost-effective and simple, and can readily be adopted by laboratories with existing MALDI MS instrumentation. The new platform and assays can be used in proteoform discovery efforts, as well as larger clinical validation studies to delineate the clinical correlations of specific proteoforms.