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.