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

Anal Chem. 2023 Jul 4;95(26):10119-10126. doi: 10.1021/acs.analchem.3c01919. Epub 2023 Jun 23.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids* / chemistry
  • Mass Spectrometry / methods
  • Protein Footprinting / methods
  • Proteins* / chemistry
  • Workflow

Substances

  • Amino Acids
  • Proteins