Traditional proteomic methods face an inherent challenge with the RAS family of GTPases (HRAS, NRAS, KRAS4A, KRAS4B). Expressed at low endogenous abundance, these four isoforms possess considerable sequence similarity, rendering the confident proteomic linkage of a given isoform with a specific mutation or post-translational modification (PTM) a formidable proposition outside of isoform-specific model cell lines. Conversely, top-down proteomic analysis (TDP), which measures intact protein molecules, can precisely identify modified protein forms, or proteoforms, and confidently localize both mutations and PTMs on each RAS isoform present. Previously, a method combining immunoprecipitation and subsequent TDP (IP-TDP) led to the identification of eleven KRAS4B proteoforms, including four mutations and two novel PTMs, within colorectal cancer cell lines and tumor samples. These initial results demonstrated the great potential for proteoform-resolved measurements to further illuminate the role of KRAS4B mutations and modifications in human cancer.

More recently, the NCI RAS Initiative has developed an enhanced RAS proteoform assay incorporating IP-TDP analysis on an Orbitrap Fusion Lumos mass spectrometer. By employing alternate antibodies, recombinant protein standards, and well-characterized model or malignant cell lines, we have obtained substantial improvements in both RAS proteoform detection and characterization, thus facilitating the identification and localization of low-abundance PTMs. Moreover, we have applied our optimized IP-TDP method to a diverse panel of malignant and RAS mutant cell lines with the goal of expanding our knowledge of the RAS proteoform landscape. In doing so, we have observed a wealth of novel RAS proteoforms, of far greater number and complexity than previously anticipated. We have also observed isoform-specific proteoform variations, most notably between KRAS4A and KRAS4B, and initial indications of proteoform context dependence, namely within a given mutation or tissue background. Our results further underscore the power of proteoform-resolved measurements and indicate that RAS-dependent signaling pathways in normal and cancer contexts may be far more complex than previously thought.

† I Ntai et. al. “Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification crosstalk.” Proc Natl Acad Sci U S A. 2018 Apr 17; 115(16):4140-4145.

Citation Format: Caroline DeHart, Kanika Sharma, Dominic Esposito, Anna Maciag, Dwight Nissley, Frank McCormick. Optimized RAS top-down proteomic assay reveals expanded proteoform landscape in malignant cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5133.