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Quantifying protein-fatty acid interactions using electrospray ionization mass spectrometry.

Lan Liu1, Elena N Kitova, John S Klassen

  • 1Alberta Ingenuity Centre for Carbohydrate Science and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.

Journal of the American Society for Mass Spectrometry
|April 8, 2011
PubMed
Summary

Direct electrospray ionization mass spectrometry (ESI-MS) can quantify bovine beta-lactoglobulin (Lg) and fatty acid (FA) interactions. Imidazole addition stabilizes complexes, improving accuracy, especially in negative ion mode, crucial for labile protein-ligand binding analysis.

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Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry
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Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

Published on: July 31, 2011

Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Mass Spectrometry

Background:

  • Bovine beta-lactoglobulin (Lg) is a major whey protein with potential applications in food and pharmaceutical industries.
  • Understanding protein-ligand interactions, such as Lg with fatty acids (FA), is crucial for characterizing its functional properties.
  • Direct electrospray ionization mass spectrometry (ESI-MS) offers a sensitive method for studying biomolecular interactions in the gas phase.

Purpose of the Study:

  • To apply direct ESI-MS to quantify the binding affinities between Lg and a series of FAs (caprylic to stearic acid).
  • To investigate the impact of in-source dissociation on the accuracy of ESI-MS measurements for these labile complexes.
  • To optimize ESI-MS conditions, including the use of additives like imidazole, to improve the detection and quantification of Lg-FA interactions.

Main Methods:

  • Direct ESI-MS was used to measure binding constants (K(a)) between Lg and various FAs.
  • Experiments were conducted in both positive and negative ion modes, with and without imidazole (10 mM).
  • Sampling conditions were adjusted to minimize in-source dissociation, and results were compared with a competitive fluorescence assay.

Main Results:

  • In-source dissociation significantly affected the accurate quantification of Lg-FA complexes, particularly protonated ions in positive mode.
  • Imidazole addition enhanced the abundance of the Lg-FA complex, improving measurement reliability.
  • ESI-MS in negative ion mode, with optimized conditions, yielded K(a) values consistent with fluorescence assays for longer-chain FAs (MA, PA, SA); however, shorter-chain FAs (CpA, CA, LA) showed significant dissociation, with CpA binding undetectable.

Conclusions:

  • Direct ESI-MS can quantify Lg-FA interactions, but in-source dissociation is a critical challenge, especially for shorter FAs and in positive ion mode.
  • Protonated Lg-FA ions are less stable than deprotonated ions, necessitating careful method optimization.
  • The use of imidazole and negative ion mode ESI-MS, coupled with minimized in-source dissociation, provides reliable quantification of labile protein-ligand complexes, as validated by the reference ligand ESI-MS assay.