Studying Protein-Ligand Interactions by Protein Denaturation and Quantitative Cross-Linking Mass Spectrometry

Anal Chem. 2023 Jun 27;95(25):9432-9436. doi: 10.1021/acs.analchem.2c04501. Epub 2023 Jun 12.

Abstract

Recently, several mass spectrometry methods have utilized protein structural stability for the quantitative study of protein-ligand engagement. These protein-denaturation approaches, which include thermal proteome profiling (TPP) and stability of proteins from rates of oxidation (SPROX), evaluate ligand-induced denaturation susceptibility changes with a MS-based readout. The different techniques of bottom-up protein-denaturation methods each have their own advantages and challenges. Here, we report the combination of protein-denaturation principles with quantitative cross-linking mass spectrometry using isobaric quantitative protein interaction reporter technologies. This method enables the evaluation of ligand-induced protein engagement through analysis of cross-link relative ratios across chemical denaturation. As a proof of concept, we found ligand-stabilized cross-linked lysine pairs in well-studied bovine serum albumin and ligand bilirubin. These links map to the known binding sites Sudlow Site I and subdomain IB. We propose that protein denaturation and qXL-MS can be combined with similar peptide-level quantification approaches, like SPROX, to increase the coverage information profiled for facilitating protein-ligand engagement efforts.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cross-Linking Reagents / chemistry
  • Ligands
  • Mass Spectrometry / methods
  • Peptides*
  • Protein Denaturation
  • Proteins* / chemistry

Substances

  • Ligands
  • Proteins
  • Peptides
  • Cross-Linking Reagents