Unambiguous characterization of PEGylation site on human amylin by two‐dimensional nuclear magnetic resonance spectroscopy

Abstract

Amylin is a 37‐residue peptide hormone, which is co‐secreted with insulin and prevents postprandial spikes in blood glycemia by slowing gastric emptying and promoting satiety. Amylin is prone to aggregate into oligomers and amyloid fibrils, which is related to the onset of type 2 diabetes, and hampers its use as a biopharmaceutical. To overcome the instability and extend its in vivo half‐life it has been proposed the conjugation of amylin with polyethylene glycol (PEG) at the HNζ or the HNα amines of Lys1. Here we used two‐dimensional nuclear magnetic resonance spectra aiming the unambiguous identification of the site of covalent modification on amylin. The coupling of PEG causes both a substantial decrease in the chemical exchange of their HN and alterations in the chemical shifts at both the HN and the neighborhood hydrocarbon groups including CHα, CHδ and CHε of Lys1. Additional analysis of chemical shifts indicates alteration in the HNα solvent accessibility of residues Cys2, Asn3, Ala5, Cys7, and Gln10, and confirmed the presence of oxidized Cys2 and Cys7. We believe that the methodology described here is a reference for the characterization of chemical coupling of a number of biopharmaceuticals.