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.

项目摘要 该提案的目的是开发简单，健壮和成本效益的质谱（MS） 蛋白质测定和平台通过结合酶促免疫测定的最佳方面 - 基于板的蛋白质 免疫亲和力捕获，MALDI-TOF MS提供了直接的MS检测。这些免疫板 - MALDI MS分析是改进的酶促免疫测定版本，其中次要记者 抗体检测步骤被MS检测取代，实现明确的蛋白质检测和 通过特定和准确的质量测量来识别。 开发新平台和测定的动力是检测和研究人类的能力 蛋白质成型 - 存在单个基因的蛋白质产物的不同分子形式。大量的 光谱法具有独特的能力，能够提供各个蛋白质成型质量的测量 原始蛋白质序列。这些蛋白质形式最好完整地检测到，这很容易通过马尔丁实现 TOF MS。一个亲和力 - 重新培养蛋白的MALDI质谱显示所有蛋白质成型化合物。 抗体涂覆的免疫板良好。可以从其信号中确定蛋白质成型的相对比率 质谱，报告为％丰度。新平台和测定将符合三个钥匙 MS蛋白质分析的促成因素：内容，成本和简单性。 在此阶段我的研究中，我们将通过原告验证免疫板摩尔迪MS MS分析来演示该平台 对于暴露了蛋白质形式的六种蛋白质生物标志物：载脂蛋白E，转铁蛋白，甲状腺素蛋白，半胱氨酸蛋白酶C，视黄醇 结合蛋白和B型亚钠肽。这些蛋白质生物标志物横跨 浓度和分子量，这将使我们能够测试平台范围，功能和 限制。使用人血浆样品将蛋白质的免疫亲和力分离 抗体涂层的96孔板，然后将洗脱捕获的蛋白质及其蛋白质成型板，并将其洗脱 在MALDI目标上发现了MS分析。实验条件将测试抗体吸附， 人血浆测定，并在MALDI靶标上洗脱。步骤数将保持在最低限度，但 足以以最低的成本获得令人满意的表现。新测定的可重复性 将进行评估，并将测定得分。 我们的目标是为一般研发市场开发免疫板毛毛细市MS平台。设计的测定 该平台将具有成本效益和简单，并且可以由现有MALDI的实验室采用 MS仪器。新平台和测定可用于蛋白质生态的发现工作，以及 较大的临床验证研究以描绘特定蛋白质相关的临床相关性。