Immunoplate-MALDI MS platform and assays

免疫板-MALDI MS 平台和检测

• 批准号: 10384628
• 负责人: DOBRIN NEDELKOV
• 金额: $ 25.62万
• 依托单位: ISOFORMIX, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10384628
• 关键词: Adopted; Adsorption; Affinity; Alzheimer' s Disease; Amino Acid Sequence; Antibodies; Apolipoprotein E; Benchmarking; Biological; Biological Assay; Brain natriuretic peptide; Clinical; Custom; Detection; Development; Digestion; Disadvantaged; Disease; Enabling Factors; Exhibits; FDA approved; Genes; Glycosylated Hemoglobin; Glycosylated hemoglobin A; Goals; Health; Human; Immobilization; Immunoassay; Individual; Laboratories; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Measurement; Measures; Molecular; Molecular Weight; Natriuretic Peptides; Pathogenesis; Pathway interactions; Peptides; Performance; Phase; Plasma; Plasma Proteins; Prealbumin; Preparation; Protein Isoforms; Proteins; Proteolysis; Proteomics; Reference Standards; Reporter; Reporting; Reproducibility; Research; Retinol Binding Proteins; Role; Sampling; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Testing; Time; Transferrin; Translating; Validation; antibody detection; apolipoprotein C-III; apolipoprotein E-4; base; carbohydrate-deficient transferrin; commercialization; cost; cost effective; design; driving force; glycosylation; improved; instrumentation; interest; microbial; post gamma-globulins; protein biomarkers; research and development; validation studies

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.

项目概要 该提案的目标是开发简单、稳健且经济高效的基于质谱 (MS) 的技术 蛋白质测定和平台结合了酶免疫测定的最佳方面 - 基于板的蛋白质 免疫亲和捕获，以及 MALDI-TOF MS 提供的简单 MS 检测。 MALDI MS 测定是酶免疫测定的改进版本，第二报告基因 抗体检测步骤被 MS 检测取代，从而实现明确的蛋白质检测和 通过对其质量进行具体而准确的测量来进行识别。 开发新平台和检测方法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。 光谱分析法具有独特的能力，可以测量各个蛋白质形式的质量，这些质量的范围从 这些蛋白质序列最好是完整检测，这可以通过 MALDI 轻松实现。 TOF MS。亲和修复蛋白的 MALDI 质谱显示了亲和力捕获的所有蛋白质形式。 抗体包被的免疫板孔中蛋白质形式的相对比例可以从它们的信号确定。 新的平台和检测方法将满足三个关键要求。 MS 蛋白质检测的促成因素：内容、成本和简单性。 在此一期研究中，我们将通过原理验证免疫板 MALDI MS 分析来演示该平台 六种表现出蛋白质形式的蛋白质生物标志物：载脂蛋白 E、转铁蛋白、运甲状腺素蛋白、半胱氨酸蛋白酶抑制剂 C、视黄醇 结合蛋白和 B 型利尿钠肽这些蛋白质生物标志物涵盖广泛。 浓度和分子量，这将使我们能够测试平台范围、功能和 利用免疫亲和力从人血浆样品中分离蛋白质是有局限性的。 抗体包被的 96 孔板，之后捕获的蛋白质及其蛋白质形式将被洗脱并 点在 MALDI 目标上进行 MS 分析 将测试抗体吸附的实验条件， 人血浆测定以及 MALDI 靶标的洗脱步骤数量将保持在最低限度。 足以以尽可能最低的成本获得令人满意的性能 新测定的可重复性。 将进行评估，并对测定进行基准测试。 我们的目标是为一般研发市场开发免疫板-MALDI MS 平台。 该平台具有成本效益且简单，可以很容易地被拥有现有 MALDI 的实验室采用 新的平台和检测方法可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，以描述特定蛋白质型的临床相关性。