Automated Specific Amino Acid Footprinting Mass Spectrometry: Repurposing an HDX Platform for Determining Reagent Feasibility

Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-L-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaaincorporated POIs hold promise for blocking undesirable POI−IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI−IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRAS G12D / RAF1, which lacks crystal structure, and generated a covalent binder to KRAS G12D . Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.

Section: Introductionmentioning

confidence: 99%

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

Ye,

Zhu,

Kong

et al. 2024

“…6,7 Specific amino acid footprinting, also referred to as covalent labeling mass spectrometry (CL-MS), is one mass spectrometry (MS)-based approach that can be employed to investigate protein HOS. 1,2,5,8−10 Specific amino acid footprinting utilizes chemical reagents to modify side-chains of a protein irreversibly based on the residue functional group chemistry, solvent accessibility, and microenvironment, 11,12 thereby increasing the mass of the protein and causing the modifications to become detectable with MS. 2,5,8 After foot-printing and digestion, liquid chromatography tandem mass spectrometry (LC-MS/MS) can be applied to locate and measure differential modification of the labeled residues. 2 Footprinting-MS offers advantages over the atomic-level techniques because footprinting requires small sample amounts (picomoles), is fast and sensitive, can be employed for a conformationally heterogeneous protein, and works in the biologically relevant liquid phase, while sometimes affording residue-level resolution.…”

Section: ■ Introductionmentioning

confidence: 99%

“…The chemical reagents utilized to footprint proteins typically react with specific functional groups or classes of groups (e.g., nucleophiles, carboxylic acids), usually enabling labeling on specific amino acids. ,,, A few examples of chemical reagents for footprinting are glycine ethyl ester (GEE), modifying aspartic and glutamic acid; N -ethylmaleimide (NEM), modifying cysteines; and diethylpyrocarbonate (DEPC) and benzoyl fluoride (BF), modifying most nucleophilic residues (Figure S1). ,,,,− The resulting modifications are generally irreversible; therefore, back-exchange and label scrambling, a concern for hydrogen–deuterium exchange (HDX)-MS, do not typically apply (although there are exceptions). ,, Despite the advantages, footprinting reagents react on a time scale of seconds to hours (depending on the reagent), a much longer time than that of protein unfolding (μs-ms time scale). ,, These side-chain modifications can induce structural perturbation, allowing further footprinting of a newly formed, non-native protein structure and leading to flawed HOS analysis. ,, One potential solution to this issue is limiting the amount of footprinting to one modification per protein, ensuring that all footprinting is on the native structure; however, restriction to a single “hit” limit not only affects the extent of modification, but also the precision, signal-to-noise ratio (S/N), and the structural resolution, especially for larger proteins. For such systems, allowing for multiple modifications on the protein using a relatively slow footprinting reagent, it is essential to evaluate the structural integrity of the protein following footprinting.…”

Section: Introductionmentioning

confidence: 99%

Evaluating Chemical Footprinting-Induced Perturbation of Protein Higher Order Structure

Wagner,

Moyle,

Wagner

et al. 2024

Self Cite

Specific amino acid footprinting mass spectrometry (MS) is an increasingly utilized method for elucidating protein higher order structure (HOS). It does this by adding to certain amino acid residues a mass tag, whose reaction extent depends on solvent accessibility and microenvironment of the protein. Unlike reactive free radicals and carbenes, these specific footprinters react slower than protein unfolding. Thus, their footprinting, under certain conditions, provokes structural changes to the protein, leading to labeling on non-native structures. It is critical to establish conditions (i.e., reagent concentrations, time of reaction) to ensure that the structure of the protein following footprinting remains native. Here, we compare the efficacy of five methods in assessing protein HOS following footprinting at the intact protein level and then further localize the perturbation at the peptide level. Three are MS-based methods that provide dose–response plot analysis, evaluation of Poisson distributions of precursor and products, and determination of the average number of modifications. These MS-based methods reliably and effectively indicate HOS perturbation at the intact protein level, whereas spectroscopic methods (circular dichroism (CD) and dynamic light scattering (DLS)) are less sensitive in monitoring subtle HOS perturbation caused by footprinting. Evaluation of HOS at the peptide level indicates regions that are sensitive to localized perturbations. Peptide-level analysis also provides higher resolution of the HOS perturbation, and we recommend using it for future footprinting studies. Overall, this work shows conclusive evidence for HOS perturbation caused by footprinting. Implementation of quality control workflows can identify conditions to avoid the perturbation, for footprinting, allowing accurate and reliable identification of protein structural changes that accompany, for example, ligand interactions, mutations, and changes in solution environment.

“…Although these experiments were performed by hand, they are apt candidates for automated specific amino acid footprinting, recently shown to expedite data collection while improving precision. 31 ■ MATERIALS AND METHODS Materials. Unless otherwise indicated, all reagents were sourced from Millipore Sigma (St. Louis, MO) and used without further purification.…”

Section: ■ Introductionmentioning

confidence: 99%

“…We acknowledge that the list is not exhaustive for all investigations with potentially novel reagents. Although these experiments were performed by hand, they are apt candidates for automated specific amino acid footprinting, recently shown to expedite data collection while improving precision …”

Section: Introductionmentioning

confidence: 99%

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

Moyle

Wagner

et al. 2023

Self Cite

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.