Deep Top-Down Proteomics Using Capillary Zone Electrophoresis-Tandem Mass Spectrometry: Identification of 5700 Proteoforms from the <i>Escherichia coli</i> Proteome

Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as a useful tool for top-down proteomics. However, its performance for deep top-down proteomics is still dramatically lower than widely used reversed-phase liquid chromatography (RPLC)-MS/MS. We present an orthogonal multidimensional separation platform that couples size exclusion chromatography (SEC) and RPLC based protein prefractionation to CZE-MS/MS for deep top-down proteomics of <i>Escherichia coli</i>. The platform generated high peak capacity (∼4000) for separation of intact proteins, leading to the identification of 5700 proteoforms from the <i>Escherichia coli</i> proteome. The data represents a 10-fold improvement in the number of proteoform identifications compared with previous CZE-MS/MS studies and represents the largest bacterial top-down proteomics data set reported to date. The performance of the CZE-MS/MS based platform is comparable to the state-of-the-art RPLC-MS/MS based systems in terms of the number of proteoform identifications and the instrument time