FLAG-KRAS4B as a Model System for KRAS4B Proteoform and PTM Evaluation by Mass Spectrometry.

Prior analysis of intact and modified protein forms (proteoforms) of KRAS4B isolated from cell lines and tumor samples by top-down mass spectrometry revealed the presence of novel posttranslational modifications (PTMs) and potential evidence of context-specific KRAS4B modifications. However, low endogenous proteoform signal resulted in ineffective characterization, making it difficult to visualize less abundant PTMs or perform follow-up PTM validation using standard proteomic workflows. The NCI RAS Initiative has developed a model system, whereby KRAS4B bearing an N-terminal FLAG tag can be stably expressed within a panel of cancer cell lines. Herein, we present a method for combining immunoprecipitation with complementary proteomic methods to directly analyze N-terminally FLAG-tagged KRAS4B proteoforms and PTMs. We provide detailed protocols for FLAG-KRAS4B purification, proteoform analysis by targeted top-down LC-MS/MS, and validation of abundant PTMs by bottom-up LC-MS/MS with example results.

A Top-Down Proteomic Assay to Evaluate KRAS4B-Compound Engagement.

Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4BG12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.

DMSO-mediated curing of several yeast prion variants involves Hsp104 expression and protein solubilization, and is decreased in several autophagy related gene (atg) mutants.

Chaperones and autophagy are components of the protein quality control system that contribute to the management of proteins that are misfolded and aggregated. Here, we use yeast prions, which are self-perpetuating aggregating proteins, as a means to understand how these protein quality control systems influence aggregate loss. Chaperones, such as Hsp104, fragment prion aggregates to generate more prion seeds for propagation. While much is known about the role of chaperones, little is known about how other quality control systems contribute to prion propagation. We show that the aprotic solvent dimethyl sulfoxide (DMSO) cures a range of [PSI+] prion variants, which are related to several misfolded aggregated conformations of the Sup35 protein. Our studies show that DMSO-mediated curing is quicker and more efficient than guanidine hydrochloride, a prion curing agent that inactivates the Hsp104 chaperone. Instead, DMSO appears to induce Hsp104 expression. Using the yTRAP system, a recently developed transcriptional reporting system for tracking protein solubility, we found that DMSO also rapidly induces the accumulation of soluble Sup35 protein, suggesting a potential link between Hsp104 expression and disassembly of Sup35 from the prion aggregate. However, DMSO-mediated curing appears to also be associated with other quality control systems. While the induction of autophagy alone does not lead to curing, we found that DMSO-mediated curing is dramatically impaired in autophagy related (atg) gene mutants, suggesting that other factors influence this DMSO mechanism of curing. Our data suggest that DMSO-mediated curing is not simply dependent upon Hsp104 overexpression alone, but may further depend upon other aspects of proteostasis.