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）作为生物医学研究中的分析工具。这证明了这一点 努力使蛋白水解的样品成为蛋白质识别和 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，最终导致数千个的深入序列确定 单个样品中的蛋白质和蛋白质同工型（自上而下方法）。大多数本机MS（NMS）应用程序 在低复杂性样品中的一组有限蛋白质和蛋白质复合物的表征的中心。 该约束归因于通过纳米电喷雾电离（Nanoesi）对直接样品输注的使用 “纯”蛋白质制剂的NMS，这受到产生足够质量和数量的需求的限制 NMS之前的靶向蛋白质。最终，这些努力是劳动，时间和成本密集。总体 TR＆D 4的目标是开发前沿分离技术，以显着促进 对人类健康基础的天然蛋白质研究，并由生物学问题驱动。这 比目前的创新，成熟和成本更高且成本更低的技术的创新，成熟和传播 可能会导致对较大的蛋白质复合物的更深入的了解。具体来说，我们将发展 i）具有额外电荷可能性的一维色谱柱色谱法 电喷雾电离期间的操作，ii）一二维纤维板分离方法 与解吸电离电离和纸张喷雾剂离子化和III）的结合，可调节毛细管 具有前所未有的选择性的电泳方法，与天然质谱兼容。强壮 具有良好发达电离技术的电离/分离专家的干部（PI：BADU），新颖 色谱固定相（PI：OLESIK），增强流体（PI：OLESIK），具有可调的智能材料 毛细管电泳分离（PI：荷兰）和NMS（PI：Wysocki）表示。这些努力利用 与两个领先的分离行业合作伙伴的现象和Sciex的合作。访问多个可溶性 蛋白质复合物（DBP 1-3A，DBP 6-10）和膜蛋白（DBP 3B-5）用作真实的测试床至 驱动器分离方法和电离技术解决了突出生物医学面临的关键障碍 该领域的研究人员。 TR＆D 4分离和电离方法协同作用，改进 在表面诱导的解离（TR＆D 1）中，离子迁移率测量（TR＆D 2）和混合方法 复杂分析（TR＆D 3），可访问来自与蛋白质相关的所有领域的更多样品 研究。结合软件开发以自动数据分析（TR＆D 5；社区信件 来自Allison，Bleiholder，Degiacomi，Marty，Prell的参与，这最终将有助于启用和精简 NMS确定蛋白质复杂结构信息。