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; 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.

项目摘要 每种蛋白质由许多不同的分子实体组成，称为蛋白质成型。这些不同的形式出现 从翻译后修改，替代性和遗传突变等。蛋白酶可以具有 不同的生物学功能，替代性可以引起疾病，并且可以用作高度特异性的生物标志物。 因此，蛋白质化分析是生物医学研究的重要领域。但是，进度很慢 因为当前的技术不足。一方面，更好的高性能蛋白质测量工具 某些应用需要更深入地进入蛋白质类型。另一方面，更快，更简单 需要工具才能使其他应用程序扩展蛋白质成型研究并通过 不是蛋白质质谱法（MS）专家的科学家。为此，该提议旨在发展 结合了分析两者所需的所有组件的独立分析设备（“墨盒”） 完整的蛋白质和蛋白水解辣椒。墨盒将包括一个基于抗体的预浓缩系统， 如果需要，固定的酶可以执行蛋白质消化，并内置底物进行蛋白质 电喷雾的电离。墨盒将允许整个蛋白质分析工作流程发生 自动在可以直接耦合到质谱仪的单个设备上。所有这些过程将 设计为无需外部抽水或其他复合系统即可进行样品处理。在AIM 1中，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的开发 电喷雾电离（底物支持的电喷雾电离或SSESI）。由 亲和力材料将洗脱到电离材料上，以使用自上而下的质量立即检测 光谱法。在AIM 2中，将将酶消化步骤集成到分析盒中以启用 检测胡椒水平的蛋白质类型。虽然蛋白酶的消化有灾难，但确实可以 通过串联质谱法进行更灵敏的检测和更好的测序。在AIM 3中，电泳 将开发方法将电离底物分离并堆叠蛋白质成型。电泳 操作将设计为几分钟之内。分离将主要设计用于减少或消除 离子抑制，同时将进行堆叠以提高检测极限。