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Permanent link (DOI): https://doi.org/10.7939/R3S17T562

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Quantifying labile protein-ligand interactions using electrospray ionization mass spectrometry Open Access

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Author or creator
El-Hawiet, Amr
Kitova, Elena N.
Liu, Lan
Klassen, John S.
Additional contributors
Subject/Keyword
Monoclonal-antibody
Complexes
Substrate
Concanavalin-A
Blood-group glycosyltransferases
Recognition
Dissociation-constants
Binding affinities
Type of item
Journal Article (Published)
Language
English
Place
Time
Description
A new electrospray ionization mass spectrometry (ES-MS) approach for quantifying protein—ligand complexes that are prone to in-source (gas-phase) dissociation is described. The method, referred to here as the reference ligand ES-MS method, is based on the direct ES-MS assay and competitive ligand binding. A reference ligand (Lref), which binds specifically to the protein (P), at the same binding site as the ligand (L) of interest, with known affinity and forms a stable protein—ligand complex in the gas phase, is added to the solution. The fraction of P bound to Lref, which is determined directly from the ES mass spectrum, is sensitive to the fraction of P bound to L in solution and enables the affinity of P for L to be determined. A mathematical framework for the implementation of the method in cases where P has one or two specific ligand binding sites is given. Affinities of two carbohydrate-binding proteins, a single chain fragment of a monoclonal antibody and the lectin concanavalin A, for monosaccharide ligands are reported and the results are shown to agree with values obtained using isothermal titration calorimetry.
Date created
2010
DOI
doi:10.7939/R3S17T562
License information
© 2010 El-Hawiet, A., Kitova, E. N., Liu, L., & Klassen, J. S. This version of this article is open access and can be downloaded and shared. The original author(s) and source must be cited.
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Citation for previous publication
El-Hawiet, A., Kitova, E. N., Liu, L., & Klassen, J. S. (2010). Quantifying labile protein-ligand interactions using electrospray ionization mass spectrometry. Journal of the American Society for Mass Spectrometry, 21(11), 1893-1899.  http://doi.org/10.1016/j.jasms.2010.07.008

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