The two KRAS isoforms, 4A and 4B, exhibit high sequence identity, differing mainly in their C-terminal “hypervariable regions”, which bear post-translational modifications (PTMs) governing their association with the plasma membrane and downstream signaling effectors. Moreover, the KRAS gene bears three “mutation hotspot” residues (Gly12, Gly13, and Gln61) represented in a high percentage of human cancers. The similarity between KRAS isoforms, in addition to HRAS and NRAS, renders linking KRAS mutations and PTMs significantly challenging by standard proteomic methods based on digestion of proteins into short peptides. However, intact modified protein forms, or proteoforms, of KRAS4A and KRAS4B can be identified and characterized by “top-down” (TD) proteomic methods, elucidating crosstalk between N-terminal mutations and C-terminal PTMs with precise molecular detail. Previously, we employed the TD approach as part of a novel workflow to precisely characterize KRAS4B proteoforms isolated from colorectal cancer cell lines and primary tumor samples by immunoprecipitation (IP) and subsequent analysis on a benchtop Orbitrap mass spectrometer (1). Here, we modified our IP-TD protocol to include KRAS4A, then applied our workflow to the characterization, quantification, and direct comparison of KRAS4A and KRAS4B proteoforms within the context of multiple cancer cell lines. We hypothesized that we would be able to identify distinct proteoform populations for each KRAS isoform, which could then be compared both within and between different contexts. We have thus far identified nine novel proteoforms of KRAS4A, which not only exhibits differential PTM to KRAS4B, but varies widely in both abundance and PTM profile across all cell lines evaluated. We have also applied IP-TD to the analysis of endogenous KRAS4A and KRAS4B proteoforms using a more powerful 21 Tesla FT-ICR mass spectrometer at the National High Magnetic Field Laboratory. The high mass resolving power, mass measurement accuracy, and spectral acquisition rate achieved at high field combined with use of efficient dissociation methods and an external fragment ion accumulator to improve signal-to-noise ratios of MS/MS spectra produced unparalleled results with respect to degree of KRAS proteoform characterization. We were able to identify up to ten new KRAS proteoforms per context, several of which appear to be mutation- or context-specific. These latest advances have broadened our understanding of KRAS biology and provided new insights into the potential roles of KRAS4A and KRAS4B proteoforms in colorectal and other cancers.

Reference

1. Ntai I, Fornelli L, DeHart CJ, Hutton JE, Doubleday PF, LeDuc RD, van Nispen AJ, Fellers RT, Whiteley G, Boja ES, Rodriguez H, Kelleher NL. 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):4140-5.

Citation Format: Caroline J. DeHart, Lissa C. Anderson, Lauren M. Adams, Luca Fornelli, Christopher L. Hendrickson, Neil L. Kelleher. Precise characterization and comparison of KRAS proteoforms by top-down mass spectrometry [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A26.