Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk

Abstract

Mutations of the gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.

The deubiquitinase inhibitor b-AP15 induces strong proteotoxic stress and mitochondrial damage

“…As a result of these advances, proteomics is now routinely used in biomarker discovery (Zhan, Li et al 2018), protein network analysis (Bennett, Rush et al 2010;Tan, Go et al 2018), cell type comparison (Gholami, Hahne et al 2013), analysis of protein stability (Leuenberger, Ganscha et al 2017;Becher, Andres-Pons et al 2018;Dai, Zhao et al 2018) and turnover (Savitski, Zinn et al 2018), as well as investigation of post-translational modifications (PTMs) (Christophorou, Castelo-Branco et al 2014;Weinert, Narita et al 2018), proteoforms (Ntai, Fornelli et al 2018) and proteogenomics (Zhu, Orre et al 2018). Arguably, one the most important applications of mass spectrometry is drug target and MOA deconvolution, in line with the chemical proteomics paradigm (Savitski, Reinhard et al 2014;Browne, Jiang et al 2018).…”

Identification and Quantification of Proteoforms by Mass Spectrometry

“…Proteoform Suite was used to quantify mouse mitochondrial proteoforms in myoblasts and differentiated myotubes and determined 129 proteoforms with statistically significant abundance changes. LFQ has also been applied in targeted approaches to quantify proteoforms from a specific proteoform family across conditions …”

In silicoapproach toward the identification of unique peptides from viral protein infection: Application to COVID-19

“…12,13 Protein-centric assays, such as intact parallel reaction monitoring or proteoform monitoring, performs well on small GTPases like KRAS, but larger proteins cannot currently be monitored with the same relative ease and accuracy. 15 For this reason, peptide-centric assays are currently the dominant proteomics approach.…”