Journal: Analytical Chemistry
Article Title: A Hit Prioritization Strategy for Compound Library Screening Using LiP-MS and Molecular Dynamics Simulations Applied to KRas G12D Inhibitors
doi: 10.1021/acs.analchem.5c03103
Figure Lengend Snippet: Limited proteolysis of KRas G12D in free and compound-bound states. (A) Crystal structure of the KRas G12D protein (PDB: 7RPZ ). The identified cleavage sites are located in the switch II loop (red), the GDP and Mg cofactors are highlighted in pink and green, respectively. (B) SDS-PAGE analysis of the limited proteolysis reactions of KRas G12D with and without compound 5 (MRTX1133), using two proteolytic enzymes (proteinase K and chymotrypsin). Lane 1, 2 – reaction with proteinase K; lane 3, 4 – reaction with chymotrypsin; lane 5 – intact KRas G12D; lane 6, 7 – proteinase K and chymotrypsin controls. There is significant protection against the KRas G12D cleavage in the presence of compound 5. (C) Intact protein LC-MS analysis of KRas G12D limited proteolysis reactions using chymotrypsin (top, orange) and proteinase K (bottom, purple) without (left) and with (right) compound 5. From the representative charge envelopes displayed, there is a noticeable absence of the protein cleavage products in the ligand-bound samples.
Article Snippet: For the limited proteolysis reactions, we used two proteolytic enzymes: chymotrypsin (TLCK treated, cat.# LS001432 , Worthington) and proteinase K (recombinant, PCR grade, Roche Diagnostics GmbH).
Techniques: SDS Page, Liquid Chromatography with Mass Spectroscopy
