Colorectal cancer (CRC) is one of the most life-threatening and prevalent forms of cancer worldwide. A better molecular-level understanding of CRC will produce novel protein biomarkers for CRC diagnosis and therapy development. Proteins play fundamental roles in modulating almost all the biological processes in cells, and different proteoforms of the same gene can have divergent biological functions. Therefore, large-scale studies of proteins in a proteoform-specific manner in CRC cells using mass spectrometry (MS)-based proteomics provide a wonderful opportunity for bettering our understanding of CRC progression and discovering new proteoform biomarkers. MS-based top-down proteomics (TDP) is ideal for the characterization of proteoforms because it measures intact proteoforms directly by employing liquid-phase separations and MS. However, TDP still faces many challenges. One of them is related to the measurement of large proteoforms (>30 kDa) from complex samples (i.e., CRC cells) due to their much wider charge state distributions and more isotopic peaks in each charge state compared to small proteoforms, leading to substantially lower signal-to-noise ratios. Many critical proteins related to CRC are larger than 30 kDa, e.g., DNA mismatch repair proteins (MSH2), EPCAM, and TP53. Therefore, the development of new TDP techniques for large proteoforms is fundamental to advance our understanding of CRC progression and biomarker discovery. Capillary zone electrophoresis (CZE)-MS is an attractive technique for TDP and has the potential to address the issues of large proteoform separation and MS detection. However, in published CZE-MS-based TDP datasets, almost all the identified proteoforms are smaller than 30 kDa. It is suspected that this phenomenon is due to sample preparation techniques and CZE-MS conditions favoring smaller proteoforms. Here, we aim to provide an improved TDP workflow for large proteoforms using optimized sample preparation and CZE-MS/MS methods.
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