Chromatographic and Electrophoretic Separations Optimized for Native MS

TR&D 4项目摘要。创新和尖端的分离和电离策略是必不可少的， 继续推进质谱（MS）作为生物医学研究中的分析工具。证明了这一点 努力使蛋白水解消化的样品成为蛋白质鉴定的更容易的应用， 量化（自下而上的方法），并通过持续发展的一维和多维 完整蛋白质的分离方法，最终导致数千种蛋白质的深入序列测定。 蛋白质和蛋白质异构体在一个单一的样品（自上而下的方法）。大多数原生MS（nMS）应用程序 集中在低复杂性样品中有限的一组蛋白质和蛋白质复合物的表征。 该限制归因于使用经由纳米电喷雾电离（nanoESI）的直接样品输注， “纯”蛋白质制剂的nMS，这受到产生足够质量和数量的蛋白质制剂的需要的限制。 在nMS之前的靶蛋白。最终，这些努力是劳动力、时间和成本密集型的。总体 TR&D 4的目标是开发尖端的分离技术，以显著提高 研究对人类健康至关重要的天然蛋白质，并由生物学问题驱动。的 技术的创新、成熟和传播，使其比目前的生产能力更高，成本更低 这一可能性将导致对更大的蛋白质复合物集合的更强理解。具体来说，我们将开发 i）一维和二维柱色谱法，具有电荷的额外可能性 ii）电喷雾电离过程中的一维和二维纤维板分离方法， 与解吸电喷雾电离和纸喷雾样电离结合，和iii）可调毛细管 电泳方法具有前所未有的选择性，与天然质谱兼容。一个强大 电离/分离专家骨干，具有完善的电离技术（PI：Badu），新颖 色谱固定相（PI：Olesik）、增强型流体（PI：Olesik）、具有可调 毛细管电泳分离（PI：Holland）和nMS（PI：Wysocki）。这些努力利用了 与Phenomenex和Sciex这两家领先的分离行业合作伙伴的合作。获得多个可溶性 蛋白复合物（DBP 1-3A，DBP 6-10）和膜蛋白（DBP 3B-5）作为一个真实的测试平台， 驱动分离方法和电离技术，解决突出的生物医学面临的关键障碍， 研究人员在该领域。TR&D 4分离和离子化方法与改进的 在表面诱导解离（TR&D 1），离子迁移率测量（TR&D 2）和混合方法中， 复合物分析（TR&D 3），可用于蛋白质相关的所有领域的更多样品 research.与软件开发相结合，实现数据分析自动化（TR&D 5;社区信函 Allison、Bleiholder、Degiacomi、Marty和Prell的参与），这将最终有助于实现和简化 蛋白质复合物结构信息的测定。

TR&D 4 Project Summary. Innovative and cutting-edge separation and ionization strategies are integral to continue advancing mass spectrometry (MS) as an analytical tool in biomedical research. This is evidenced by efforts to make proteolytically digested samples a more accessible application for protein identification and quantification (bottom-up approach) and through the continued development of one- and multi-dimensional separation methods for intact proteins, which ultimately lead to in-depth sequence determination of thousands of proteins and protein isoforms within a single sample (top-down approach). Most native MS (nMS) applications center on the characterization of a limited set of proteins and protein complexes in samples of low complexity. This constraint is attributed to the use of direct sample infusion via nano electrospray ionization (nanoESI) for nMS of 'pure' protein preparations, which is limited by the need to generate a sufficient quality and quantity of the targeted protein prior to nMS. Ultimately these efforts are labor, time, and cost intensive. The overarching goal of TR&D 4 is to develop cutting-edge separation technologies in order to significantly advance research in native proteins fundamental to human health, and driven by biological problems. The innovation, maturation, and dissemination of technologies with a higher throughput and lower cost than currently possible will lead to a stronger understanding of a larger set of protein complexes. Specifically, we will develop i) one- and two-dimensional column chromatographic methods with the additional possibility for charge manipulation during electrospray ionization, ii) one- and two-dimensional fiber plate separation methods in conjunction with desorption electrospray ionization and paper spray-like ionization, and iii) tunable capillary electrophoresis methods with unprecedented selectivity, compatible with native mass spectrometry. A strong cadre of ionization/ separation experts with well-developed ionization techniques (PI: Badu), novel chromatographic stationary phases (PI: Olesik), enhanced fluids (PI: Olesik), smart materials with tunable capillary electrophoresis separations (PI: Holland), and nMS (PI: Wysocki) is represented. These efforts leverage collaborations with Phenomenex and Sciex, two leading separation industry partners. Access to multiple soluble protein complexes (DBP 1-3A, DBP 6-10) and membrane proteins (DBP 3B-5) serve as an authentic test-bed to drive separation methods and ionization techniques that address critical barriers faced by prominent biomedical researchers in the field. The TR&D 4 separation and ionization methodologies are synergistic with improvements in surface-induced dissociation (TR&D 1), ion mobility measurements (TR&D 2), and hybrid methods for complex-down analysis (TR&D 3), accessible for a greater number of samples from all areas of protein related research. In combination with software developments to automate data analysis (TR&D 5; letters in Community Engagement from Allison, Bleiholder, Degiacomi, Marty, Prell), this will ultimately help to enable and streamline the determination of protein complex structure information by nMS.