Oct 9, 2019 – MSDG Monthly Meeting

One fifth of human cancers contain mutations activating the Ras GTPases, but no targeted therapies are yet available. Many effectors of the Ras pathway and potential targets remain underexplored and we have limited understanding of numerous paralogs of Ras effectors and other redundancies that contribute to Ras tumor growth. Here, we produce a multiomic map of the Ras function in lung adenocarcinoma. We use affinity purification mass spectrometry (AP/MS) to generate a protein-protein interaction (PPI) network. From this network, we constructed a sgRNA library covering 120 Ras pathway genes and screened for genetic interactions (GIs) by CRISPR dual knockout. We find new effectors of Ras-driven cellular function, and over 250 synthetic lethal GIs, presenting new candidate single and combination therapeutic targets. We identify important synergies between Ras and Rap signaling and between their downstream effector pathways. Our recent studies have used selective covalent inhibitors of a KRAS-G12C allele from the Shokat lab to address global signaling changes by shotgun proteomics, using the Bruker timsTOF. I will present the latest technical details for our data and also outline our informatics pipeline, which provides critical validation of our MS findings. These discoveries illustrate the power of multiomic interaction-mapping approach and may be applied to analyze a range of human diseases or other biological systems.

Confidence in mass spectrometry measurements is key for advancing drug discovery projects. Bruker provides a comprehensive line of ESI and MALDI TOF instruments as well industry leading ultrahigh resolution MRMS systems and the evolutionary timsTOF Pro. The timsTOF Pro with PASEF relies on a unique dual Trapped Ion Mobility Spectrometry (TIMS) front end to provide nearly 100% duty cycle with no loss of ions. When combined with the Parallel Accumulation Serial Fragmentation (PASEF) method, the timsTOF Pro with PASEF enables sequencing speeds in excess of 100 Hz, and significant improvements in sensitivity. Powered by TIMS, the instrument provides separation for simple isomers and conformers and stands as an integrated part of 4D omics workflows because of the accurate and reproducible collisional cross-sections (CCS) values for all peptides. Recently, the TIMS technology has been applied to host cell protein (HCP) analysis of mAbs with record speed and depth. Newly announced diaPASEF enables more ways to accurately quantify proteins. The presentation will show how the technology can benefit HCP analysis and OMICS to step up the research to the next level.