Chromatographic and Electrophoretic Separations Optimized for Native MS

R&D 4项目总结。创新和尖端的分离和电离策略是 继续将质谱学(MS)作为生物医学研究的分析工具。这一点从以下几个方面得到了证明 努力使经蛋白质消化的样品更容易用于蛋白质鉴定和 量化(自下而上的方法)，并通过一维和多维的持续发展 完整蛋白质的分离方法，最终导致对数千种蛋白质的深入序列测定 在单个样本中分析蛋白质和蛋白质异构体(自上而下的方法)。大多数本地MS(NMS)应用程序 集中在低复杂性样本中有限的一组蛋白质和蛋白质复合体的特征。 这一限制归因于通过纳米电喷雾电离(NanESI)直接样品输注用于 NMS的“纯”蛋白质制剂，这受到需要产生足够质量和数量的 NMS之前的靶蛋白。归根结底，这些工作是劳动力、时间和成本密集型的。最重要的是 研发4的目标是开发尖端分离技术，以显著推进 对天然蛋白质的研究对人类健康至关重要，并受到生物学问题的推动。这个 以比当前更高的吞吐量和更低的成本进行技术创新、成熟和传播 可能会导致对更大一组蛋白质复合体的更深入的理解。具体来说，我们将发展 一)增加收费可能性的一维和二维柱层析方法 电喷雾电离过程中的操作，II)一维和二维纤维板分离方法 结合脱附电喷雾电离和纸喷雾状电离，以及iii)可调毛细管 电泳法具有前所未有的选择性，与天然质谱学兼容。一个坚强的人 拥有先进电离技术的电离/分离专家队伍(PI：BADU)，新颖 色谱固定相(PI：Olesik)、增强型流体(PI：Olesik)、可调智能材料 毛细管电泳法(PI：Holland)和NMS(PI：Wysocki)。这些努力利用了 与分离行业的两个领先合作伙伴Phenomeex和Sciex合作。获得多个可溶的 蛋白质复合体(DBP1-3A，DBP6-10)和膜蛋白(DBP3B-5)是真正的试验台 驱动分离方法和电离技术，解决了著名生物医学面临的关键障碍 该领域的研究人员。T&D 4分离和电离方法是协同的，并有改进 在表面诱导解离(tr&d1)、离子迁移率测量(tr&d2)和混合方法中 复杂向下分析(TR&D 3)，可用于所有相关蛋白质领域的更多样本 研究。与软件开发相结合，实现数据分析自动化(R&D 5；社区信函 来自Allison、Bleihold、Degiacomi、Marty、Prell的合作)，这最终将有助于实现和简化 蛋白质复合体结构信息的NMS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.