Oncogenic mutations of KRAS are found in ~22% of human cancers and result in constitutive activation of downstream pathways. These pathways promote cell survival, proliferation and tumorigenesis, affecting cancer progression and treatment outcome. Here, we describe a new workflow that can quantify mutation-specific consequences of KRAS structure and biochemistry, such as linked changes in post-translational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down proteomics to map and quantify intact KRAS4b proteoforms with or without the Gly13Asp mutation (G13D). This seminar will focus on results from isogenic colorectal cancer (CRC) cell lines and then transition to patient CRC tumors with matching KRAS genotypes. In two cellular models, a direct link between the knock-out of the mutant G13D allele and nitrosylation of cysteine 118 of the remaining wild type KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually expressed in cancer tissue and identified major differences in C-terminal carboxymethylation known to be critical for membrane association. Switching from targeted to discovery modes, we will survey results from use of top-down proteomics in systems biology and for biomarker discovery in organ transplantation. Finally, a new “native” for top-down proteomics (i.e., “Native Proteomics”) will be introduced, where the complete composition of endogenous protein complexes can be determined (Nat. Chem. Biol. 2017 Nov 13. doi: 10.1038/nchembio.2515).
Dr. Ronghu Wu (email@example.com)