Advancing the Reproducibility and Repeatability of Capillary Zone Electrophoresis-Mass Spectrometry-Based Top-Down Proteomics by an Improved Capillary Coating Procedure

Capillary zone electrophoresis (CZE)-mass spectrometry (MS) has attracted tremendous attention in top-down proteomics (TDP). However, its reproducibility and long-term repeatability for TDP remain concerns, most likely due to capillary coating. Here, we present an improved procedure for making linear polyacrylamide (LPA) coating, the most widely used coating in CE-MS-based proteomics, to boost the reproducibility and long-term repeatability of CZE-MS-based TDP. We focused on the step of degassing the polymerization solution, a critical step for achieving consistent LPA coating quality. The CZE-MS system using LPA-coated capillaries prepared with the optimal degassing procedure produced excellent reproducibility and repeatability for proteoform analysis. The 210 CZE-MS runs of three protein samples (a standard protein mixture, an E. coli cell lysate, and a HeLa cell lysate) across 200 h of instrument time with two MS instruments demonstrated the reliability of our conclusion. The optimal condition produced relative standard deviations (RSDs) of less than 2.6% in the migration time of proteoforms across dozens of CZE-MS runs without migration time alignment. CZE-MS/MS analysis of a HeLa whole cell lysate identified 274 ± 20 proteoforms (RSD ∼ 7%) and 146 ± 10 proteins (RSD ∼ 7%) across 10 successive runs and yielded consistent proteoform intensities. The coating procedure can be easily adopted by other research groups, as evidenced by our 2025 CE-MS summer school data. All results demonstrate that CZE-MS using the optimal LPA-coating procedure is ready for broad adoption to enable reproducible and repeatable measurements of proteoforms in complex samples.