Intact Proteoform Analysis by Capillary Electrophoresis–Mass Spectrometry. Are We There Yet?
Noah Gould, Qianjie Wang, Jeffrey N. Agar, Jennifer S. Brodbelt, Daoyang Chen, Kellye A. Cupp‐Sutton, Elena Domínguez‐Vega, Fei Fang, Marianne Fillet, Matthew S. Fischer, Attila Gáspár, Ying Ge, Marie‐Jia Gou, Michal Greguš, Christoph Gstöttner, Narmin Hamidli, Amanda Helms, Md Amin Hossain, Kyle J. Juetten, Neil L. Kelleher, Tobias Kraus, Eli J. Larson, J. Scott Mellors, Cynthia Nagy, Christian Neusüß, Erin A. Redman, Jasmin Schairer, Si Wu, Tian Xu, Zhitao Zhao, Guijie Zhu, Alexander R. Ivanov, Kevin Jooß, Liangliang Sun
Abstract
Mass spectrometry (MS)-based top-down proteomics (TDP) has emerged as a powerful tool for characterizing proteoforms to advance both fundamental and translational research. TDP requires high-efficiency liquid-phase separation, high-resolution MS, and tandem MS. Capillary zone electrophoresis (CZE)-MS has been proposed as a promising analytical technique for protein analysis decades ago because of its unique and valuable features, including high separation efficiency and high detection sensitivity. However, CZE-MS has not been widely adopted by the proteomics community, mainly due to concerns with its robustness and reproducibility. Here, we hypothesized that CZE-MS is sufficiently robust and reproducible for broad adoption due to the continued efforts of the community over the last three decades. In this work, for the first time, research teams from around the world validated the robustness, repeatability, and reproducibility of CZE-MS for TDP in both simple and complex model proteoform mixtures employing a full spectrum of commercially available capillary electrophoresis (CE)-MS interfaces, instrumentation, and compared CZE-MS performance with state-of-the-art liquid chromatography (LC)-MS methods. This study offers the research community an informative resource of ready-to-use experimental CE-MS techniques and a better understanding of the CZE-MS approach and its potential in TDP, accelerating the broad adoption of CZE-MS in proteoform research.