Site-Specific characterization of peptide-polymer conjugates in various stoichiometries by MALDI-Tandem mass spectrometry

PEGylation, covalent linking of polyethylene glycol (PEG) chains, is one of the most common ways for enhancing the drug properties of biopharmaceuticals. The linkage of PEG chains improves the pharmacokinetic properties of therapeutic biomolecules by increasing their hydrodynamic radius and also shields them against immune system. The chemical composition and structural features of biomolecule-PEG conjugates should be well-characterized in a fast and easy methods to project their therapeutic efficiency. For this purpose, mass spectrometry is the best analytical technique to analyze biopharmaceuticals in very low amounts with high sensitivity. Also, the stoichiometric ratio of species composing biomolecule-PEG conjugates can be easily calculated by using data obtained from mass spectrometric analysis. Moreover, tandem mass spectrometry (MS²) analysis, controlled breakdown of the isolated ions in the gas phase, also gives detailed information on chemical structures of species. The binding site of PEG chains on biomolecules can be determined by evaluating data obtained from MS² analysis. In the study, angiotensin II peptide was PEGylated by using PEG chains with Mw=600 containing modified acid halide end-groups. MALDI-MS and MALDI-MS² analyses were performed for obtaining data on determination of binding sites and stoichiometric ratio of angiotensin II and PEG chains for conjugates.