title: Monitoring ligand modulation of protein-protein interactions by mass spectrometry: estrogen receptor alpha-SRC1.
abstract: Many drugs and chemicals exert their biological effect by modulating protein-protein interactions. In vitro approaches to characterize these mechanisms are often based on indirect measurements (e.g., fluorescence). Here, we used mass spectrometry (MS) to directly monitor the effect of small-molecule ligands on the binding of a coactivator peptide (SRC1) by the human estrogen receptor alpha ligand binding domain (hERalpha LBD). Nanoelectrospray mass spectrometry (nanoESI-MS) and high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking were employed to follow these processes. The chemical cross-linking protocol used prior to high-mass MALDI analysis allows detection of intact noncovalent complexes. The binding of intact hERalpha LBD homodimer with two coactivator peptides was detected with nanoESI-MS and high-mass MALDI-MS only in the presence of an agonist ligand. Furthermore, high-mass MALDI-MS revealed an increase of the homodimer abundance after incubating the receptor with a ligand, independent of the ligand character (i.e., agonist, antagonist). The binding characteristics of the compounds tested by MS correlate very well with their biological activity reported by cell-based assays. High-mass MALDI appears to be an efficient and simple tool for directly monitoring ligand regulation mechanisms involved in protein-protein interactions. Furthermore, the combination of both MS methods allows identifying and characterizing endocrine-disrupting compounds or new drug compounds in an efficient way.
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland.
authors.forename: Cédric
authors.initials: C
authors.lastname: Bovet
authors.forename: Marc
authors.initials: M
authors.lastname: Ruff
authors.forename: Sylvia
authors.initials: S
authors.lastname: Eiler
authors.forename: Florence
authors.initials: F
authors.lastname: Granger
authors.forename: Ryan
authors.initials: R
authors.lastname: Wenzel
authors.forename: Alexis
authors.initials: A
authors.lastname: Nazabal
authors.forename: Dino
authors.initials: D
authors.lastname: Moras
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
mesh_terms: D000595:Amino Acid Sequence; D019281:Dimerization; D052244:Endocrine Disruptors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010600:Pharmacology; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D052244:Endocrine Disruptors; D047628:Estrogen Receptor alpha; D008024:Ligands; D010455:Peptides