| Autor: |
Moyle AB; Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States., Wagner ND; Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States., Wagner WJ; Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States., Cheng M; Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States., Gross ML; Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States. |
| Jazyk: |
angličtina |
| Zdroj: |
Analytical chemistry [Anal Chem] 2023 Jul 04; Vol. 95 (26), pp. 10119-10126. Date of Electronic Publication: 2023 Jun 23. |
| DOI: |
10.1021/acs.analchem.3c01919 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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