Intact quantitation and evaluation of a PEG-glycosulfopeptide as a therapeutic P-selectin antagonist

Peptide-based therapeutics are recognized as potent and selective molecules but are often limited by short circulating half-lives, instability towards enzymatic degradation, and immunogenicity. To address these limitations and improve their pharmacological properties, peptides are commonly modified by the covalent attachment of polyethylene glycol (PEG). However, the large molecular weight and polydispersity of PEG chains complicate the interpretation of the full structure of PEGylated peptide therapeutics using standard analytical techniques. Here, we developed a mass spectrometric-based workflow in negative ion mode to identify and quantify GSnP-6, a P-selectin antagonist, with a linear 10 kDa PEG (PEG10) attached at the N-terminus of the glycopeptide. Intact mass analysis with multiple microscans allowed accurate measurements of precursor ions in complex biological mixtures with baseline resolution. Utilizing stepped collision energies improved sequence coverage and enabled identification of key amino acid modifications. We show the utility of this approach in evaluating the properties of PEG10-GSnP-6 in vitro and in vivo. Inhibitory capacity was preserved while extending the half-life of this glycopeptide, as shown by the reduction of P-selectin/PSGL-1 binding. By sustaining effective circulating concentrations, PEG conjugation of a P-selectin glycopeptide antagonist represents a promising therapeutic strategy to target diseases linked to inflammatory processes.