Project Summary It has been estimated that 145,600 people were diagnosed with colorectal cancer (CRC) and 51,020 deaths were predicted due to this disease in 2019 (https://seer.cancer.gov/statfacts/html/colorect.html). A better understanding of CRC at the molecular level will certainly lead to therapies that are more effective. The genome- level and transcriptome-level information cannot accurately reflect the protein-level information because post-transcriptional regulation can modulate protein expression and because post-translational modifications (PTMs) can influence protein function. Quantitative proteomic studies of CRC are vital. Many bottom-up proteomics studies have been completed on CRC cells and tumors, but limited information on proteoforms have been acquired due to low protein sequence coverages typically obtained from bottom-up proteomics. Different proteoforms from the same gene can have drastically different functions. We hypothesize that large-scale and quantitative top-down proteomics of human CRC cells and tumors will provide new insights into CRC, leading to better therapies. In this proposal, we will develop new analytical tools to boost the sensitivity and scale of top-down proteomics. The new tools will enable large-scale and quantitative top-down proteomics of CRC cells before and after metastasis as well as CRC tumors from patients with Lynch Syndrome. Results from this proposal are extremely important. The novel analytical tools will boost the sensitivity of top-down proteomics by tenfold and will be particularly useful for the proteomics community for large-scale top-down proteomics of mass-limited samples. Quantitative top-down proteomics of CRC cells before and after metastasis will generate an unprecedented resource for the cancer biology community to gain new insights into CRC metastasis. Quantitative top-down proteomics of the Lynch Syndrome tissues will elucidate the roles played by mutations and functions of DNA mismatch repair genes in Lynch Syndrome at the proteoform level.