Combining Separation, Digestion, and Ionization on a Mass Spectrometry Cartridge to Enable Biomedical Research on Proteoforms

在质谱柱上结合分离、消化和电离，以实现蛋白质形式的生物医学研究

• 批准号: 10637225
• 负责人: Nicholas E Manicke
• 金额: $ 34.01万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-25 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10637225
• 关键词: Affinity; Antibodies; Biological; Biological Process; Biology; Biomedical Research; Cell Culture Techniques; Chemicals; Chemistry; Complex; Coupled; Crowding; DNA Sequence Alteration; Detection; Development; Devices; Diagnostic; Diagnostic tests; Digestion; Disadvantaged; Disease; Disease Marker; Disease Progression; Electrophoresis; Electrospray Ionization; Engineering; Goals; Human Biology; Immobilization; Immobilized Enzymes; Indiana; Ions; Liquid substance; Mass Spectrum Analysis; Measurement; Measures; Membrane; Methods; Molecular; Neurofibromin 2; Paper; Parents; Pepsin A; Peptide Hydrolases; Peptides; Performance; Porosity; Post-Translational Protein Processing; Process; Protein Analysis; Proteins; Proteome; Pump; RNA Splicing; Research; Research Personnel; Resolution; Role; Sampling; Scientist; Serum; Single Nucleotide Polymorphism; Specialist; Specificity; Speed; System; Technology; Time; Trypsin; Universities; Variant; Western Blotting; Work; accurate diagnostics; chemical property; cost; design; detection limit; disease prognostic; exome sequencing; experimental study; frontier; high throughput analysis; human disease; human genome sequencing; improved; instrument; ionization; mass spectrometer; metabolomics; novel strategies; physical property; portability; protein purification; scale up; specific biomarkers; tandem mass spectrometry; targeted treatment; tool

Project Summary Each protein consists of numerous different molecular entities, termed proteoforms. These different forms arise from post-translation modifications, alternative, and genetic mutations among others. Proteoforms can have different biological function, alternations can cause disease, and they can serve as highly specific biomarkers. Proteoform analysis is, therefore, an important frontier in biomedical research. Progress is slow, however, because current technology is inadequate. On one hand, better high-performance protein measurement tools are needed for some applications to reach ever deeper into the proteoform. On the other hand, faster and simpler tools are needed for other applications to scale up proteoform research and increase participation in the field by scientists who are not protein mass spectrometry (MS) specialists. To that end, this proposal aims to develop self-contained analytical devices (‘cartridges’) that combines all of the components necessary to analyze both intact proteins and proteolytic peptides. The cartridge will include an antibody-based preconcentration system, an immobilized enzyme to perform protein digestion if desired, and a built-in substrate to perform protein ionization by electrospray. The cartridge will allow the entirety of the protein analysis workflow to occur automatically on a single device that can be directly coupled to the mass spectrometer. All of these processes will be designed to work without external pumping or other complex systems for sample handling. In Aim 1, a device will be developed that combines affinity-based protein purification with a porous material to perform electrospray ionization (substrate-supported electrospray ionization or ssESI). Proteoforms captured by the affinity material will be eluted onto the ionization material for immediate detection using top-down mass spectrometry. In Aim 2, an enzymatic digestion step will be integrated into the analysis cartridge to enable detection of proteoforms at the peptide level. While digestion of proteoforms has disadvantages, it does enable more sensitive detection and better sequencing by tandem mass spectrometry. In Aim 3, electrophoretic methods will be developed to separate and stack proteoforms on the ionization substrate. The electrophoretic manipulations will be designed to occur in minutes. Separations will be mainly designed to reduce or eliminate ion suppression, while stacking will be done to improve detection limits.

项目概要 每种蛋白质由许多不同的分子实体组成，称为蛋白质形式，这些不同的形式出现。 来自翻译后修饰、替代和基因突变等。 不同的生物学功能、改变可能导致疾病，并且它们可以作为高度特异性的生物标志物。 因此，蛋白质组分析是生物医学研究的一个重要前沿领域，但进展缓慢。 因为目前的技术还不够，更好的高性能蛋白质测量工具。 另一方面，某些应用程序需要更快、更简单地深入了解蛋白质形式。 其他应用需要工具来扩大蛋白质型研究并增加该领域的参与 为此，本提案旨在开发非蛋白质质谱 (MS) 专家的科学家。 独立的分析设备（“盒”），结合了分析两者所需的所有组件 完整的蛋白质和蛋白水解肽。该盒将包括基于抗体的预浓缩系统， 如果需要的话，可以使用固定化酶来进行蛋白质消化，还可以使用内置底物来进行蛋白质消化 通过电喷雾进行电离，该盒将允许整个蛋白质分析工作流程发生。 所有这些过程都将在可直接耦合到质谱仪的单个设备上自动完成。 In Aim 1 是一种无需外部泵或其他复杂样品处理系统即可工作的设备。 将开发将基于亲和力的蛋白质纯化与多孔材料相结合以执行 电喷雾电离（底物支持的电喷雾电离或 ssESI）。 亲和材料将被洗脱到电离材料上，以便使用自上而下的质量立即检测 在目标 2 中，酶消化步骤将集成到分析盒中，以实现 在肽水平上检测蛋白质形式虽然蛋白质形式的消化有缺点，但它确实能够实现。 通过串联质谱实现更灵敏的检测和更好的测序 在目标 3 中，电泳。 将开发在电离基质上分离和堆叠蛋白质形式的方法。 操作将在几分钟内发生，分离的主要目的是减少或消除。 离子抑制，同时将进行堆叠以提高检测限。