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Deciphering Polymer Interactions in Bioconjugates with Different Architectures by Structural Analysis via

Prisca Hamm1, Marc D Driessen2,3, Niklas Hauptstein1

  • 1Universität Würzburg, Institute for Pharmacy and Food Chemistry, Am Hubland, 97074, Würzburg, Germany.

Angewandte Chemie (International Ed. in English)
|January 9, 2025
PubMed
Summary

Limited proteolysis mass spectrometry (LiP-MS) reveals how polymers like polyethylene glycol (PEG) interact with interferon-alpha2a (IFN). This technique enhances understanding of transient protein-polymer interactions for bioconjugate development.

Keywords:
15N labelingInterferon alpha 2aLimited proteolysisbioconjugatesquantitative proteomics

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Area of Science:

  • Biochemistry
  • Proteomics
  • Polymer Chemistry

Background:

  • Therapeutic proteins are often modified with polymers to improve their pharmacokinetic properties.
  • Understanding the specific interactions between polymers and proteins is crucial for designing effective bioconjugates.
  • Current methods often lack detailed descriptions of these polymer-protein interactions.

Purpose of the Study:

  • To investigate and structurally characterize the interactions between interferon-alpha2a (IFN) and various polymers using limited proteolysis mass spectrometry (LiP-MS).
  • To compare the interaction profiles of polyethylene glycol (PEG), linear polyglycerol (LPG), and poly(2-oxazoline) (POX) with IFN-α2a.
  • To elucidate the influence of different polymer linkers (BCN and DBCO) on protein-polymer interactions.

Main Methods:

  • Limited proteolysis mass spectrometry (LiP-MS) was employed for time-resolved quantification of trypsin cleavage dynamics on IFN-α2a.
  • Site-specific bioconjugation of 10 kDa polymers (PEG, LPG, POX) to azide-functionalized IFN-α2a using cyclooctyne linkers (BCN/DBCO).
  • All-atom molecular dynamics simulations were performed to analyze transient protein-polymer interactions.

Main Results:

  • LiP-MS revealed distinct interaction profiles: PEG and LPG showed similar interactions with IFN-α2a, while POX exhibited reduced surface interaction.
  • Molecular dynamics simulations confirmed transient ( < 50 ns) and distinct interaction profiles for PEG, LPG, and POX with IFN.
  • IFN-polymer conjugates formed via the BCN linker displayed homogeneous cleavage dynamics, suggesting a more conserved IFN structure compared to DBCO-linked conjugates.

Conclusions:

  • Time-resolved LiP-MS is a powerful tool for quantifying cleavage events and enhancing structural understanding of transient protein-polymer interactions.
  • The choice of polymer and linker significantly influences the interaction profile and structural dynamics of bioconjugates.
  • These findings provide valuable insights for the rational design of novel protein-polymer bioconjugates with modulated pharmacokinetics.