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.