Method for Distinguishing Specific from Nonspecific Protein−Ligand Complexes in Nanoelectrospray Ionization Mass Spectrometry
Abstract
A new methodology for distinguishing between specific and nonspecific protein-ligand complexes in nanoelectrospray ionization mass spectrometry (nanoES-MS) is described. The method involves the addition of an appropriate reference protein (P(ref)), which does not bind specifically to any of the solution components, to the nanoES solution containing the protein(s) and ligand(s) of interest. The occurrence of nonspecific protein-ligand binding is monitored by the appearance of nonspecific (P(ref) + ligand) complexes in the nanoES mass spectrum. Furthermore, the fraction of P(ref) undergoing nonspecific ligand binding provides a quantitative measure of the contribution of nonspecific binding to the measured intensities of protein and specific protein-ligand complexes. As a result, errors introduced into protein-ligand association constants, K(assoc), as determined with nanoES-MS, by nonspecific ligand binding can be corrected. The principal assumptions on which this methodology is based, namely, that the fraction of proteins and protein complexes that engage in nonspecific ligand binding during the nanoES process is determined by the number of free ligand molecules in the offspring droplets leading to gaseous ions and is independent of the size and structure of the protein or protein complex, are shown to be generally valid. The application of the method for the determination of K(assoc) for two protein-carbohydrate complexes, under conditions where nonspecific ligand binding is prevalent, is demonstrated.
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Informed estimations of relative binding affinities can be made by fitting the intensity ratio of bound and unbound protein as a function of ligand concentration.
“Nevertheless, informed estimations can be made about relative binding affinities by fitting the intensity ratio of bound and unbound protein as a function of ligand concentration21,72.”
From Native metabolomics identifies the rivulariapeptolide family of protease inhibitors (2022) · cites this paper as [72]
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“For the latter, careful analysis is required as nonspecific association may occur during the ionization process for some systems, especially when the analyte concentrations are high and/or the binding affinities low [84, 85].”
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- Authors
- Sun, J., Kitova, E. N., Wang, W., & Klassen, J. S.
- Affiliations
- University of Alberta (CA)
- Corresponding author
- Klassen, John S.
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2006-04-01
- Volume
- 78
- Issue
- 9
- Pages
- 3010-3018
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- OpenAlex
- W2004681646
- Cited by
- 160
- References
- 14
- Citations by year
- 2026: 6 · 2025: 4 · 2024: 5 · 2023: 3 · 2022: 7 · 2021: 10 · 2020: 9 · 2019: 6 · 2018: 7 · 2017: 12 · 2016: 12 · 2015: 11 · 2014: 15 · 2013: 12 · 2012: 7
- FWCI
- 3.65
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Engineering › Electrical and Electronic Engineering › Electrohydrodynamics and Fluid Dynamics
- Keywords
- protein-ligand complex, reference proteins, binding constant, protein-carbohydrate complex, binding specificity, free ligand concentration
- MeSH terms
- Binding Sites, Carbohydrate Conformation, Carbohydrate Sequence, Carbohydrates, Ligands, Molecular Sequence Data, Proteins, Sensitivity and Specificity, Spectrometry, Mass, Electrospray Ionization, Nanotechnology
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