Reproducible Capillary Electrophoresis-Mass Spectrometry-Based Top-Down Proteomics of Complex Proteomes Enabled by an...

Capillary zone electrophoresis-mass spectrometry (CZE-MS) has become a powerful tool for top-down proteomics (TDP). Capillaries are commonly coated with neutral or cationic polymers to reduce protein adsorption in CZE-MS. We recently introduced a cationic polymer coating [poly(acrylamide-co-(3-acrylamidopropyl) trimethylammonium chloride (PAMAPTAC)] to CZE-MS-based TDP. Here, we further improved the coating procedure to enhance the ease and repeatability of the cationic coating preparation by employing 2,2'-azobisisobutyronitrile (AIBN) as the radical initiator and methanol as the solvent. The new coating method not only simplifies the coating process but is also user-friendly. Different laboratory members were able to reproduce this type of cationic coating with consistent CZE-MS performance. We evaluated the performance of CZE-MS with the cationic coating in analyzing various complex biological samples (nanoparticle protein corona, histone, and an Escherichia coli cell lysate) and assessed its long-term reproducibility with an E. coli cell lysate for 35 runs (over 40 h) of continuous measurement. The CZE-MS system produced reproducible measurements for all the samples regarding separation profiles, proteoform migration time (MT), and proteoform intensity. For example, the system enabled the detection of large proteoforms (26-35 kDa) from an E. coli sample with consistent MT, relative standard deviations (RSDs) <7% without MT correction and 2.1% after MT alignment. We expect that the APTAC coating will further advance CZE-MS-based TDP for broad and large-scale applications.