Chromatographic and Electrophoretic Separations Optimized for Native MS

针对非变性 MS 优化的色谱和电泳分离

• 批准号: 10441402
• 负责人: Susan V. Olesik
• 金额: $ 12.9万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10441402
• 关键词: Address; Adsorption; Affinity Chromatography; Area; Beds; Biological; Biomedical Research; Blood capillaries; Capillary Electrophoresis; Charge; Collaborations; Column Chromatography; Complex; Complex Mixtures; Consumption; Data Analyses; Detection; Development; Dimensions; Dissociation; Electrolytes; Electrospray Ionization; Fiber; Goals; Health; Human; Hybrids; Hydrophobic Interactions; Infusion Technique; Infusion procedures; Ion Exchange; Ion-Exchange Chromatography Procedure; Ions; Lead; Letters; Liquid substance; Mass Spectrum Analysis; Measurement; Membrane Proteins; Methodology; Methods; Molecular Sieve Chromatography; Paper; Peptides; Phase; Phospholipids; Preparation; Protein Isoforms; Proteins; Research; Research Personnel; Resolution; Resources; Risk; Sampling; Sequence Determination; Set protein; Signal Transduction; Sodium Chloride; Specificity; Structure; Surface; System; Techniques; Technology; Testing; Time; analytical tool; base; community engagement; cost; design; improved; industry partner; innovation; interest; ion mobility; ionization; ionization technique; nano-electrospray; novel; protein complex; protein folding; protein structure; software development; structural biology; tool; two-dimensional

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.

TR&D 4 项目摘要。创新和尖端的分离和电离策略是不可或缺的 继续推进质谱 (MS) 作为生物医学研究的分析工具。这可以证明 努力使蛋白水解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，最终导致数千种蛋白质的深入序列测定 单个样品中的蛋白质和蛋白质亚型（自上而下的方法）。大多数本机 MS (nMS) 应用程序 重点是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。 这一限制归因于通过纳米电喷雾电离 (nanoESI) 直接注入样品 “纯”蛋白质制剂的 nMS，受到生成足够质量和数量的蛋白质的需要的限制 nMS 之前的目标蛋白。最终，这些努力是劳动、时间和成本密集型的​​。首要的 TR&D 4 的目标是开发尖端分离技术，以显着推进 由生物问题驱动的对人类健康至关重要的天然蛋白质的研究。这 比目前更高吞吐量和更低成本的技术创新、成熟和传播 可能将导致对更多蛋白质复合物有更深入的了解。具体来说，我们将开发 i) 一维和二维柱色谱方法，具有额外的电荷可能性 电喷雾电离过程中的操作，ii）一维和二维纤维板分离方法 与解吸电喷雾电离和纸喷雾式电离结合，以及 iii) 可调谐毛细管 电泳方法具有前所未有的选择性，与天然质谱法兼容。一个强大的 拥有成熟电离技术的电离/分离专家骨干（PI：Badu），新颖 色谱固定相（PI：Olesik）、增强液（PI：Olesik）、可调智能材料 代表了毛细管电泳分离（PI：Holland）和nMS（PI：Wysocki）。这些努力利用 与两个领先的分离行业合作伙伴 Phenomenex 和 Sciex 合作。获得多种可溶性 蛋白质复合物（DBP 1-3A、DBP 6-10）和膜蛋白（DBP 3B-5）可作为真实的测试平台 推动分离方法和电离技术，解决著名生物医学面临的关键障碍 该领域的研究人员。 TR&D 4 分离和电离方法与改进具有协同作用 表面诱导解离 (TR&D 1)、离子淌度测量 (TR&D 2) 以及混合方法 复合物下调分析 (TR&D 3)，可用于蛋白质相关所有领域的更多样品 研究。与软件开发相结合，实现数据分析自动化（TR&D 5；社区信件 Allison、Bleiholder、Degiacomi、Marty、Prell 的参与），这最终将有助于实现和简化 通过 nMS 测定蛋白质复杂结构信息。