Workflow for Validating Specific Amino Acid Footprinting Reagents for Protein Higher Order Structure Elucidation

Abstract

Protein footprinting mass spectrometry probes protein higher order structure and dynamics by labeling amino acid side-chains or backbone amides as a function of solvent accessibility. One category of footprinting uses residue-specific, irreversible covalent modifications, affording flexibility of sample processing for bottom-up analysis. Although several specific amino acid footprinting technologies are becoming established in structural proteomics, there remains a need to assess fundamental properties of new reagents before their application. Often, footprinting reagents are applied to complex or novel protein systems soon after their discovery and sometimes without a thorough investigation of potential downsides of the reagent. In this work, we assemble and test a validation workflow that utilizes cyclic peptides and a model protein to characterize benzoyl fluoride, a recently published, next-generation nucleophile footprinter. The workflow includes the characterization of potential side-chain reactive groups, reaction “quench” efficacies, reagent considerations and caveats (e.g., buffer pH), residue-specific kinetics compared to those of established reagents, and protein-wide characterization of modification sites with considerations for proteolysis. The proposed workflow serves as a starting point for improved footprinting reagent discovery, validation, and introduction, the aspects of which we recommend before applying to unknown protein systems.

Evaluating Chemical Footprinting-Induced Perturbation of Protein Higher Order Structure

“…For BF footprinting, the dose–response plot displays linearity up to 20 and 7.5 equiv with k values of 0.176 ± 0.006 mM –1 s –1 and 0.125 ± 0.009 mM –1 s –1 for myoglobin and lysozyme, respectively (Figure B). The rate constants are larger for BF compared to those for DEPC, which is likely explained by the higher reactivity rate of Lys and Tyr with BF and the slightly different reaction conditions for the two reagents.…”

“…BF is more hydrophobic than DEPC (logP value for BF is 2.08 and for DEPC is 0.75) and is more likely to footprint in hydrophobic, less solvent-exposed regions of the protein. Another explanation is that the rate constants for each modifiable residue are different for DEPC and BF, as reported by Moyle et al The footprinting conditions necessary to provoke HOS perturbation should depend upon the innate reactivity of the modifiable residues as adjusted by the surrounding microenvironment. Most importantly, these data indicate that every footprinter can differentially perturb the HOS at various regions of the protein.…”

“…A second issue is that the rate of modification is assumed to be constant for each reactive residue, but this assumption is unlikely to hold because the inherent reactivities of each residue are different, and surrounding microenvironments and solvent accessibilities are distinct. 26 The Poisson distribution fitting analysis may be more suitable for footprinting reagents that only have one reactive residue (such as NEM footprinting of Cys residues). In such cases, the reactivity will be affected by local solvent accessibility and microenvironment (versus footprinting with DEPC/BF where there are many reactive residues).…”

“…DEPC has become a standard footprinting reagent, wellcharacterized by the Vachet group, 1,2,9,11,20,21,24,25 whereas BF is a relatively new footprinter from our lab. 14,26 The Vachet group has spearheaded efforts to investigate footprinting-induced perturbation with DEPC, as they are the primary investigating lab involved in the development of dose−response plot analysis, while also utilizing some CD analysis. 2,9,24 More recently, Kirsch and Vachet used binomial distribution modeling to determine an appropriate amount of footprinter reagent without inducing perturbation, showing slight changes in protein HOS upon implementing extensive footprinting conditions.…”

Carbene Footprinting Directs Design of Genetically Encoded Proximity-Reactive Protein Binders

“…To address this challenge, we propose to introduce protein footprinting − a powerful structural MS techniqueinto the GECB workflow to rationally direct PrUaa anchorage. Previously, protein footprinting has been frequently used to map PPI interfaces and to identify spatially proximal residues as binding sites of PPIs of interest. , Even though it only provides low-resolution, residue-level structural information in comparison to high-resolution crystallographic data generated by cryo-electron microscopy (cryo-EM), X-ray diffraction (XRD) and nuclear magnetic resonance (NMR), − footprinting offers unique advantages including high throughput, sensitivity, minimal protein consumption and independence from crystallography. , Among various footprinting methods, diazirine-based carbene footprinting is appealing. , The carbene donor of choice in this study is trifluoromethylaryl diazirine (TFMAD).…”