Pilot Evaluation of the Long-Term Reproducibility of Capillary Zone Electrophoresis–Tandem Mass Spectrometry for Top-Down Proteomics of a Complex Proteome Sample

Mass spectrometry (MS)-based top-down
proteomics (TDP) has revolutionized
biological research by measuring intact proteoforms in cells, tissues,
and biofluids. Capillary zone electrophoresis–tandem MS (CZE-MS/MS)
is a valuable technique for TDP, offering a high peak capacity and
sensitivity for proteoform separation and detection. However, the
long-term reproducibility of CZE-MS/MS in TDP remains unstudied, which
is a crucial aspect for large-scale studies. This work investigated
the long-term qualitative and quantitative reproducibility of CZE-MS/MS
for TDP for the first time, focusing on a yeast cell lysate. Over
1000 proteoforms were identified per run across 62 runs using one
linear polyacrylamide (LPA)-coated separation capillary, highlighting
the robustness of the CZE-MS/MS technique. However, substantial decreases
in proteoform intensity and identification were observed after some
initial runs due to proteoform adsorption onto the capillary inner
wall. To address this issue, we developed an efficient capillary cleanup
procedure using diluted ammonium hydroxide, achieving high qualitative
and quantitative reproducibility for the yeast sample across at least
23 runs. The data underscore the capability of CZE-MS/MS for large-scale
quantitative TDP of complex samples, signaling its readiness for deployment
in broad biological applications. The MS RAW files were deposited
in ProteomeXchange Consortium with the data set identifier of PXD046651.