McCool, Elijah N.Lubeckyj, RacheleShen, XiaojingKou, QiangLiu, XiaowenSun, Liangliang2021-08-092021-08-092018-10-24McCool, E. N., Lubeckyj, R., Shen, X., Kou, Q., Liu, X., & Sun, L. (2018). Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry. JoVE (Journal of Visualized Experiments), 140, e58644. https://doi.org/10.3791/586441940-087Xhttps://hdl.handle.net/1805/26403Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.en-USChemistryIssue 140Top-down proteomicscapillary zone electrophoresiselectrospray ionizationtandem mass spectrometryproteoformEscherichia coliArticle