Journal of The American Society for Mass Spectrometry

, Volume 28, Issue 9, pp 1863–1875 | Cite as

Insight into Signal Response of Protein Ions in Native ESI-MS from the Analysis of Model Mixtures of Covalently Linked Protein Oligomers

  • Katharina Root
  • Yves Wittwer
  • Konstantin Barylyuk
  • Ulrike Anders
  • Renato Zenobi
Research Article

Abstract

Native ESI-MS is increasingly used for quantitative analysis of biomolecular interactions. In such analyses, peak intensity ratios measured in mass spectra are treated as abundance ratios of the respective molecules in solution. While signal intensities of similar-size analytes, such as a protein and its complex with a small molecule, can be directly compared, significant distortions of the peak ratio due to unequal signal response of analytes impede the application of this approach for large oligomeric biomolecular complexes. We use a model system based on concatenated maltose binding protein units (MBPn, n = 1, 2, 3) to systematically study the behavior of protein mixtures in ESI-MS. The MBP concatamers differ from each other only by their mass while the chemical composition and other properties remain identical. We used native ESI-MS to analyze model mixtures of MBP oligomers, including equimolar mixtures of two proteins, as well as binary mixtures containing different fractions of the individual components. Pronounced deviation from a linear dependence of the signal intensity with concentration was observed for all binary mixtures investigated. While equimolar mixtures showed linear signal dependence at low concentrations, distinct ion suppression was observed above 20 μM. We systematically studied factors that are most often used in the literature to explain the origin of suppression effects. Implications of this effect for quantifying protein–protein binding affinity by native ESI-MS are discussed in general and demonstrated for an example of an anti-MBP antibody with its ligand, MBP.

Graphical Abstract

Keywords

Native ESIMS Response factors Concatenated protein 

Supplementary material

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Copyright information

© American Society for Mass Spectrometry 2017

Authors and Affiliations

  • Katharina Root
    • 1
  • Yves Wittwer
    • 1
  • Konstantin Barylyuk
    • 1
    • 2
  • Ulrike Anders
    • 1
  • Renato Zenobi
    • 1
  1. 1.Department of Chemistry and Applied BiosciencesETH ZurichZurichSwitzerland
  2. 2.Department of BiochemistryUniversity of CambridgeCambridgeUK

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