Intact mass spectrometry screening to optimize hydroxyl radical dose for protein footprinting.
Biochem Biophys Res Commun
; 671: 343-349, 2023 09 03.
Article
en En
| MEDLINE
| ID: mdl-37329657
ABSTRACT
Hydroxyl radical protein footprinting (HRPF) using synchrotron radiation is a well-validated method to assess protein structure in the native solution state. In this method, X-ray radiolysis of water generates hydroxyl radicals that can react with solvent accessible side chains of proteins, with mass spectrometry used to detect the resulting labeled products. An ideal footprinting dose provides sufficient labeling to measure the structure but not so much as to influence the results. The optimization of hydroxyl radical dose is typically performed using an indirect Alexa488 fluorescence assay sensitive to hydroxyl radical concentration, but full evaluation of the experiment's outcome relies upon bottom-up liquid chromatography mass spectrometry (LC-MS) measurements to directly determine sites and extent of oxidative labeling at the peptide and protein level. A direct evaluation of the extent of labeling to provide direct and absolute measurements of dose and "safe" dose ranges in terms of, for example, average numbers of labels per protein, would provide immediate feedback on experimental outcomes prior to embarking on detailed LC-MS analyses. To this end, we describe an approach to integrate intact MS screening of labeled samples immediately following exposure, along with metrics to quantify the extent of observed labeling from the intact mass spectra. Intact MS results on the model protein lysozyme were evaluated in the context of Alexa488 assay results and a bottom-up LC-MS analysis of the same samples. This approach provides a basis for placing delivered hydroxyl radical dose metrics on firmer technical grounds for synchrotron X-ray footprinting of proteins, with explicit parameters to increase the likelihood of a productive experimental outcome. Further, the method directs approaches to provide absolute and direct dosimetry for all types of labeling for protein footprinting.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Radical Hidroxilo
/
Huella de Proteína
Tipo de estudio:
Diagnostic_studies
/
Screening_studies
Idioma:
En
Revista:
Biochem Biophys Res Commun
Año:
2023
Tipo del documento:
Article