An expanded RAS proteoform landscape provided by top-down analysis of malignant cell lines

Top-down mass spectrometry (TDMS), which analyzes intact and modified protein forms (proteoforms), can confidently identify and precisely localize mutations or post-translational modifications (PTMs) while maintaining the primary sequence. This provides unparalleled molecular detail, particularly in the case of the RAS GTPases (HRAS, KRAS4A, KRAS4B, NRAS), which are the most frequently mutated proteins in human cancer. Their high sequence identity, labile PTMs, and low endogenous abundance have all provided formidable challenges to standard peptide-based proteomic methods. However, by directly analyzing immunoprecipitated RAS proteoforms via targeted TDMS (IP-TDMS), the NCI RAS Initiative has identified novel RAS PTMs and context-specific RAS proteoforms which provide new insight into RAS-dependent signaling mechanisms.

Previously, we developed an optimized RAS IP-TDMS assay on an Orbitrap Fusion Lumos mass spectrometer. By analyzing recombinant, FLAG-tagged, and endogenous versions of each RAS isoform, we identified chromatographic gradients, proteoform charge states, and isoform-specific targeted MS2 parameters to facilitate optimal proteoform characterization. However, after an expanded analysis of malignant cell lines employing these optimized parameters, many endogenous RAS proteoforms still could not be successfully characterized due to their low relative abundance and certain expected RAS proteoforms (HRAS, KRAS4A) were still not observed. To address this, we further optimized our RAS IP-TDMS assay on an Orbitrap Exploris 480 mass spectrometer. Here, we have substantially expanded the RAS proteoform landscape by performing IP-TDMS analysis on fourteen malignant cell lines (NCI-60 and NCI Ras Initiative validated) using previously optimized parameters and both instrument platforms.

In doing so, we identified an unanticipated wealth of novel HRAS, KRAS4A, KRAS4B, and NRAS proteoforms, the majority of which differed markedly from the established literature. We also observed further evidence of context-dependent RAS proteoforms, including apparent tissue-, isoform- and mutation-specific modifications. Our results underscore the ability of proteoform-resolved measurements to improve our understanding of RAS-dependent signaling while emphasizing that much work remains to be done to elucidate the role of RAS PTMs and proteoforms in oncogenesis.