Wang, ZheYu, DahangCupp-Sutton, Kellye A.Liu, XiaowenSmith, KennethWu, Si2021-08-092021-08-092020-08-28Wang, Z., Yu, D., Cupp-Sutton, K. A., Liu, X., Smith, K., & Wu, S. (2020). Development of an Online 2D Ultrahigh-Pressure Nano-LC System for High-pH and Low-pH Reversed Phase Separation in Top-Down Proteomics. Analytical Chemistry, 92(19), 12774–12777. https://doi.org/10.1021/acs.analchem.0c033950003-2700https://hdl.handle.net/1805/26352The development of novel high-resolution separation techniques is crucial for advancing the complex sample analysis necessary for high-throughput top-down proteomics. Recently, our group developed an offline 2D high-pH RPLC/low-pH RPLC separation method and demonstrated good orthogonality between these two RPLC formats. Specifically, ultrahigh-pressure long capillary column RPLC separation has been applied as the second dimensional low-pH RPLC separation for the improvement of separation resolution. To further improve the throughput and sensitivity of the offline approach, we developed an online 2D ultrahigh-pressure nano-LC system for high-pH and low-pH RPLC separations in top-down proteomics. An online microtrap column with a dilution setup was used to collect eluted proteins from the first dimension high-pH separation and inject the fractions for ultrahigh-pressure long capillary column low-pH RPLC separation in the second dimension. This automatic platform enables the characterization of 1000+ intact proteoforms from 5 μg of intact E. coli cell lysate in 10 online-collected fractions. Here, we have demonstrated that our online 2D pH RP/RPLC system coupled with top-down proteomics holds the potential for deep proteome characterization of mass-limited samples because it allows the identification of hundreds of intact proteoforms from complex biological samples at low microgram sample amounts.en-USEscherichia coliNanotechnologyProteomicsChromatographyArticle