Deciphering Proteoform Landscape of Mammary Carcinoma by Top-Down Proteomics.

Defining the proteoform landscape of breast cancer can provide unique insights into the signaling pathways driving disease progression. While bottom-up proteomics has been utilized to profile breast cancer, it lacks the ability to capture intact proteoforms that may underpin the disease. Top-down proteomics is ideally suited to characterize intact proteoforms; however, most top-down proteomics studies have been limited to low molecular weight (MW) proteins (<50 kDa). Herein, we employed a two-dimensional (2D) liquid chromatography combining size exclusion chromatography (SEC) with reverse phase chromatography (RPC) followed by high-resolution mass spectrometry (MS) to expand the coverage for high MW proteoforms. Using this 2D-SEC-RPC-MS approach, we observed a 5-fold increase in the detection of high MW proteoforms (>50 kDa) compared to the conventional 1D-RPC-MS. SEC separation significantly enhanced the detection of high MW proteoforms (>104 kDa), including intermediate filament proteins, vimentin and keratins. Based on accurate mass measurements and MS/MS data, we identified 775 proteoforms from both TFA and HEPES extracts and detected PTMs, such as acetylation, glutathionylation, and myristoylation. Pathway analysis uncovered many proteoforms involved in processes dysregulated in cancer progression. Overall, our findings illustrate the power of top-down proteomics in defining the proteoform landscape of breast carcinoma.