Fig. 5: Application of intact protein LC-MS to oncology targets.
Covalent inhibitors sotorasib and ibrutinib targeting the undruggable KRASG12C and BTK protein, correspondingly, were chosen to test the scope of the methods presented here. Representative raw and deconvoluted spectra (inset) with drug-related mass shift for each of the protein targets are shown. Together (a) and (b) confirm the expected sotorasib-related mass shift ( + 560.6 Da) using a commercially sourced KRASG12C (green). In addition, the mechanism of action (Decision#1, D1) and METE (D2) were confirmed using an in-house purified (c), (d) GNP (blue) and (e), (f) GDP-bound (purple) versions of KRAS, confirming sotorasib selectively binds the inactive GDP-KRASG12C, thereby trapping the protein in its inactive conformation. (g), (h) Ibrutinib covalently binds to BTK ( + 438.6 Da) (pink) confirming the MoA and METE criteria. The phosphorylated proteoforms of BTK are delineated with a bracket. Notably, this analysis shows that unmodified BTK as well as BTK phospoproteoforms with 1, 2, and 3 phosphorylation sites fully bind ibrutinib. The unbound mass of BTK (average mass: 77512.5 Da) corresponds to an unknown +88 Da modified proteoform of BTK that cannot bind ibrutinib. Total ion current is represented as a.u. (arbitrary units).
