Activation State-Selective Kinase Inhibitor Assay Based on Ion Mobility-Mass Spectrometry

Abstract

The discovery of activation state dependent kinase inhibitors, which bind specifically to the inactive conformation of the protein, is considered to be a promising pathway to improved cancer treatments. Identifying such inhibitors is challenging, however, because they can have Kd values similar to molecules known to inhibit kinase function by interacting with the active form. Further, while inhibitor induced changes within the kinase tertiary structure are significant, few technologies are able to correctly assign inhibitor binding modes in a high-throughput fashion based exclusively on protein-inhibitor complex formation and changes in local protein structure. We have developed a new assay, using ion mobility-mass spectrometry, capable of both rapidly detecting inhibitor binding and classifying the resultant kinase binding modes. Here, we demonstrate the ability of our approach to classify a broad set of kinase inhibitors, using micrograms of protein, without the need for protein modification or tagging.

Effects of Multidentate Metal Interactions on the Structure of Collisionally Activated Proteins: Insights from Ion Mobility Spectrometry and Molecular Dynamics Simulations

“…28 Our findings suggest that comparative measurements will yield reliable results only when electrospraying different samples in solutions that generate exactly the same adduction patterns. For small proteins the adduction status is usually apparent in the mass spectra.…”

“…On the other hand, it is also possible to deliberately expose gaseous proteins to collisional excitation for probing structural and dynamic features. 19,[26][27][28][29] For example, it has been proposed that CIU "fingerprints" can help distinguish between different modes of inhibitor binding. 28 In the case of protein complexes, CIU and CID go hand in hand.…”

“…Moderately energetic collisions with background gas are required for proper desolvation, 25 whereas higher activation levels cause collisioninduced unfolding (CIU) as well as collision-induced dissociation (CID) of noncovalent complexes. 19,[26][27][28][29] Experimentalists often strive to avoid CIU and CID. On the other hand, it is also possible to deliberately expose gaseous proteins to collisional excitation for probing structural and dynamic features.…”

Bacterial Electron Transfer Chains Primed by Proteomics

“…When calibrated with proteins of known structures, collected data allow the calculation of the average collisional cross section of a protein (in Å 2 ) providing important 3D structural information. In addition, these data enable the calculation of dissociation constants of binding partners as well as the revelation of subtle conformational changes and ligand binding-induced unfolding patterns (Laganowsky et al, 2014;Niu & Ruotolo, 2015;Rabuck et al, 2013;Stojko et al, 2015). Thus, by IM-MS information is gathered on the 3D structure and on the dynamics of a protein.…”

Towards integrative structural mass spectrometry: Benefits from hybrid approaches

“…By acting like ''gas phase capillary electrophoresis'', ion mobility brings an additional dimension of separation, virtually increasing the peak capacity of the instrument allowing not only clean-up of a spectrum but also the detection of subtle conformational changes [90] or differences in unfolding patterns upon ligand binding [91,92]. Moreover, CCSs constitute structural constraints that can be very useful for molecular modeling purposes [69,[93][94][95].…”