Therapeutic proteins are commonly conjugated with polymers to modulate their pharmacokinetics but often lack a description of the polymer-protein interaction. We deployed limited proteolysis mass spectrometry (LiP-MS) to reveal the interaction of polyethylene glycol (PEG) and PEG alternative polymers with interferon-α2a (IFN). Target conjugates were digested with the specific protease trypsin and a "heavy" 15N-IFN wild type (IFN-WT) for time-resolved quantification of the cleavage dynamics. Interactions between IFN-α2a and its high-affinity receptor were detailed by LiP-MS. Then, 10 kDa polymers of PEG, linear polyglycerol (LPG), and poly(2-oxazoline) (POX) with two different cyclooctyne linkers (BCN/DBCO) were used for site-specific bioconjugation to azide functionalized IFN-α2a. Tryptic events at each cleavage site and in different structural environments (loops/helices) were compared. PEG and LPG were similar, and POX showed a reduced interaction profile with the IFN-α2a surface. All-atom molecular dynamics simulations of IFN-DBCO-polymer conjugates revealed distinct and transient (below 50 ns) protein-interaction profiles for PEG, LPG, and POX. Cleavage dynamics of IFN-polymer conjugates from the BCN handle were homogeneous, pointing to a more conserved IFN structure than DBCO-polymer conjugates. In summary, time-resolved LiP-MS for quantification of cleavage events enhances the structural understanding of transient IFN-polymer interactions, which may be extended to other bioconjugate types.
Keywords: 15N labeling; Interferon alpha 2a; Limited proteolysis; bioconjugates; quantitative proteomics.
© 2025 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.