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

项目摘要 每种蛋白质由许多不同的分子实体组成，称为蛋白质形式。这些不同的形式出现了 翻译后修改、替代和基因突变等。蛋白质类可以有 不同的生物功能，变化会导致疾病，它们可以作为高度特异的生物标志物。 因此，蛋白质组分析是生物医学研究的重要前沿。然而，进展缓慢， 因为目前的技术是不够的。一方面，更好的高性能蛋白质测量工具 是某些应用程序深入蛋白质形式所必需的。另一方面，更快、更简单 其他应用程序需要工具来扩大蛋白质形式研究并通过以下方式增加该领域的参与度 不是蛋白质质谱仪(MS)专家的科学家。为此，这项提案旨在发展 自给自足的分析设备(‘试剂盒’)，结合了分析两者所需的所有成分 完整的蛋白质和蛋白水解肽。该试剂盒将包括基于抗体的预浓缩系统， 如果需要，用于执行蛋白质消化的固定化酶，以及用于执行蛋白质的内置底物 电喷雾电离。试剂盒将允许进行整个蛋白质分析工作流程 自动安装在可直接连接到质谱仪的单个设备上。所有这些过程都将 设计为无需外部泵或其他复杂的样品处理系统即可工作。在《目标1》中，一种装置 将开发一种将基于亲和力的蛋白质纯化与多孔性材料相结合来执行 电喷雾电离(衬底支持的电喷雾电离或ssESI)。捕获的蛋白质组 亲和物质将被洗脱到电离材料上，使用自上而下的质量立即进行检测 光谱分析。在目标2中，酶消化步骤将集成到分析盒中，以使 在多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 更灵敏的检测和更好的串联质谱仪测序。在AIM 3中，电泳法 将开发在电离底物上分离和堆积蛋白质形式的方法。电泳法 操作将被设计为在几分钟内发生。分离将主要是为了减少或消除 离子抑制，同时将进行堆积以提高检测下限。

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.