High-Mass Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Absolute Quantitation of Noncovalent Protein–Protein Binding Interactions
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Open access
Date
2021-08-10Type
- Journal Article
Abstract
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a robust and powerful tool for studying biomacromolecules and their interactions. However, quantitative detection of high-mass analytes (kDa to MDa range) remains challenging for MALDI-MS. Herein, we successfully used commercially available purified proteins (β-galactosidase and BSA) as internal standards for high-mass MALDI-MS analysis and achieved absolute quantification of several high-mass analytes. We systematically evaluated four sample deposition methods, and using the sandwich deposition method with saturated sinapinic acid as the top layer, we performed a robust quantitative analysis by high-mass MALDI-MS. Combined with chemical cross-linking, this quantitative strategy was further used to evaluate the affinity of protein–protein interactions (PPIs), specifically of two soluble protein receptors (interleukin 1 receptor and interleukin 2 receptor) and two membrane protein receptors (rhodopsin and angiotensin 2 receptor 1) with their interaction partners. The measured dissociation constants of the protein complexes formed were between 10 nM and 5 μM. We expect this high-throughput, rapid method, which does not require labeling or immobilization of any of the interaction partners, to become a viable alternative to traditional biophysical methods for studying PPIs. Show more
Permanent link
https://doi.org/10.3929/ethz-b-000502791Publication status
publishedExternal links
Journal / series
Analytical ChemistryVolume
Pages / Article No.
Publisher
American Chemical SocietySubject
Mass spectrometry; Peptides and proteins; Layers; Deposition; ReceptorsOrganisational unit
03430 - Zenobi, Renato / Zenobi, Renato
Funding
178765 - Soft ionization mass spectrometry for studying noncovalent interactions (SNF)
Related publications and datasets
Is supplemented by: https://doi.org/10.3929/ethz-b-000495980
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