Leveraging Gas-Phase Fragmentation Pathways for Improved Identification and Selective Detection of Targets Modified by Covalent Probes

Abstract

The recent approval of covalent inhibitors for multiple clinical indications has reignited enthusiasm for this class of drugs. As interest in covalent drugs has increased, so too has the need for analytical platforms that can leverage their mechanism-of-action to characterize modified protein targets. Here we describe novel gas phase dissociation pathways which yield predictable fragment ions during MS/MS of inhibitor-modified peptides. We find that these dissociation pathways are common to numerous cysteine-directed probes as well as the covalent drugs, Ibrutinib and Neratinib. We leverage the predictable nature of these fragment ions to improve the confidence of peptide sequence assignment in proteomic analyses and explore their potential use in selective mass spectrometry-based assays.

A Chemoproteomic Strategy for Direct and Proteome-Wide Covalent Inhibitor Target-Site Identification

“…Motivated by the growing clinical impact of CKIs, juxtaposed with key concerns related to potential toxicities as described above, we sought to develop CITe-Id as a robust chemoproteomic strategy that would go beyond protein-level identification of targets, and provide a residue-level direct readout of concentration-dependent covalent binding of a given CKI. We previously described specific gas-phase fragmentation pathways for peptides covalently modified by cysteine-directed, irreversible inhibitors and clinical drugs, 20 including our recently described CDK7/12/13 inhibitor, THZ1 5 (Figure 1A). Building on these insights, we first elaborated THZ1 with a desthiobiotin affinity handle (THZ1-DTB, Figure 1A (1)) and then used Western blot to confirm concentration-dependent labeling and enrichment of CDK7 (Supporting Information, SI, Figure S1A).…”

“…CITe-Id directly quantifies covalent probe binding with site-level resolution. (A) Structures of: THZ1 described by Kwiatkowski et al; 5 desthiobiotinylated analog (THZ1-DTB); and THZ531 described by Zhang et al 6 (B) Workflow of CITe-Id using THZ1/THZ1-DTB: (1) Cell lysates were treated with THZ1/THZ1-DTB in a competition format, (2) protease digestion was carried out before streptavidin enrichment to provide a highly enriched pool of CKI-labeled peptides, (3) samples were encoded with iTRAQ stable isotope reagents to enable mass spectrometry quantification of concentration-dependent binding at individual cysteine residues, (4) online, automated RP-SAX-RP chromatography 23 was tailored for the increased hydrophobicity of labeled peptides and allowed for ready adjustment of fractionation depth, (5) preprocessing of MS/MS spectra following our previous framework 20 improved identification of cysteine residues covalently bound by THZ1- DTB. Competitive binding dose–response curves generated from iTRAQ reporter ions were used to distinguish concentration-dependent/independent binding of CKIs.…”

“…We recently demonstrated that cysteine-directed probes and covalent drugs share common gas-phase dissociation path-ways. 20 Pertinent to the limitations noted above, the predictable nature of these fragment ions can be used to improve peptide sequence assignment including the specific site of covalent modification. Here, we build on these results to establish a new chemoproteomic platform that leverages affinity-tagged analogs of pharmacologic CKIs for the biochemical enrichment of targets, along with tunable peptide fractionation and custom spectral processing to identify inhibitor target sites.…”

mzStudio: A Dynamic Digital Canvas for User-Driven Interrogation of Mass Spectrometry Data

“…Furthermore, mzStudio supports custom spectral processing algorithms ( Supplementary Figure S1 illustrates a custom processing routine written in Python); this capability enables in-depth exploration of surprising or novel gas-phase fragmentation behavior. We used these tools to significantly improve identification rates for peptides modified with cysteine-directed covalent drugs and other chemical probes [ 17 ]. With mzStudio, researchers can add, refine, or create entirely new spectral pre-processing routines (for examples, see the example_processing_scripts folder in the Github repository), submit MS/MS data to multiple search algorithms, and assess the impact both qualitatively (improved utilization or accounting of fragment ions) and quantitatively (individual peptide score).…”

Evaluation and Optimization of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Protein Interactomes

“…More recently, modifications to cleavable biotin linkers have enabled a diverse set of chemically-cleavable reagents which provide the advantage of decreased sample processing times due to faster cleavage conditions and small residual modifications on conjugated peptides, a highly desirable property that can affect the ability to determine the identities of these peptides 12,13 . Different chemical moieties have been implemented in the structure of cleavable linkers [14][15][16] .…”