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

Abstract

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.

Top-Down Proteomics Analysis of Picogram-Level Complex Samples Using Spray-Capillary-Based Capillary Electrophoresis–Mass Spectrometry

“…However, we noted some run-to-run variation in separation with the spray-capillary. This may be attributed to protein stacking on the inner capillary wall over multiple consecutive runs, leading to a reversal in EOF that slows proteoform migration and broadens peaks, as suggested by the Sun group . The robustness and lifetime of the spray-capillary CE–MS analysis can be improved with proper and frequent regeneration.…”