Chromatographic and Electrophoretic Separations Optimized for Native MS

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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.