Abstract A016: Expanded RAS proteoform landscape in malignant cell lines revealed by top-down mass spectrometry

Abstract The RAS family of GTPases (HRAS, KRAS4A, KRAS4B, NRAS) provide a formidable challenge to standard proteomic workflows. Linking key N-terminal mutations (e.g., G12C, G13D) with C-terminal post-translational modifications (PTMs) on a given RAS isoform by proteolytic digestion and peptide-based analysis is further complicated by their high sequence identity, low endogenous abundance, and labile PTMs. Conversely, top-down mass spectrometry (TDMS), which measures intact and modified protein forms (proteoforms), can precisely identify and confidently localize mutations and PTMs within each RAS isoform sequence. Moreover, by combining immunoprecipitation with TDMS (IP-TDMS), endogenous RAS proteoforms can be isolated to near-homogeneity from malignant cell lines, detected at high signal-to-noise ratios via selected ion monitoring, and targeted for optimized MS2-based characterization to provide molecular detail simply unachievable by other proteomic methods. The NCI RAS Initiative has developed an optimized RAS proteoform assay incorporating IP-TDMS analysis on an Orbitrap Fusion Lumos mass spectrometer. We initially employed recombinant versions of each RAS isoform to determine the optimal targeted MS2 fragmentation (tMS2) parameters for sequence characterization and localization of endogenous PTMs. We next isolated FLAG-tagged KRAS4B -WT, -G12D, and -G12C expression constructs from HeLa cells to refine our liquid chromatography and tMS2 parameters while targeting stoichiometrically abundant KRAS4B proteoforms within each population. We then isolated FLAG-tagged HRAS-, KRAS4A-, KRAS4B-, and NRAS-G12V proteins from HeLa or Panc1 cells for further refinement of isoform-specific tMS2 parameters with emphasis on capturing unusual or labile PTMs (e.g. palmitoylation). This allowed us to visualize and directly compare the most abundant RAS isoform-specific proteoform populations within these cellular contexts. Complementary peptide-based analyses were performed on all samples to validate RAS isoform-specific PTM landscapes and facilitate detection of their respective endogenous versions. Finally, we applied our optimized IP-TDMS workflow to a panel of well-characterized malignant and RAS mutant cell lines with the goal of expanding our knowledge of the endogenous RAS proteoform landscape. In doing so, we identified a wealth of novel HRAS, KRAS4A, KRAS4B, and NRAS proteoforms bearing stoichiometrically abundant PTMs differing markedly from the established literature. We also performed direct comparisons between isoform- and mutation-specific RAS proteoforms, revealing clear indications of context dependence. These initial results further underscore the potential for top-down proteomics to broaden our understanding of RAS-dependent signaling within a range of cancer contexts. They also reveal that extensive investigations remain to be performed to elucidate the roles of RAS PTMs and proteoforms in normal and oncogenic signaling. Citation Format: Caroline J. DeHart, Robert A. D'Ippolito, Kanika Sharma, Nicole Fer, Brian Smith, Mackenzie Meyer, Scott Eury, Abigail Neish, Katie Powell, Vanessa Wall, William Burgan, Dominic Esposito, Anna E. Maciag, Frank McCormick, Dwight V. Nissley. Expanded RAS proteoform landscape in malignant cell lines revealed by top-down mass spectrometry [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A016.