Structural characterization of protein–polymer conjugates. I. Assessing heterogeneity of a small PEGylated protein and mapping conjugation sites using ion exchange chromatography and top-down tandem mass spectrometry

PEGylated proteins are a rapidly growing class of biopharmaceutical products, but their analytical characterization remains a formidable problem due to the extreme heterogeneity of these species. While significant advances have been made in recent years in this field due to integration of mass spectrometry in the analytical workflow, quick identification of PEGylation sites remains an unmet goal, particularly if several isoforms of the protein–polymer conjugate are present in the sample. To achieve this objective, a new method is developed, which utilizes a combination of ion exchange chromatography and top-down mass spectrometry consisting of two consecutive fragmentation steps (MS3) to identify various conjugates. The method is tested with a complex mixture of products of ubiquitin conjugation with 5 kDa PEG.

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► Characterization of PEGylated proteins is a formidable problem due to their heterogeneity.
► Quick identification of PEGylation sites is a particularly challenging task.
► Combination of ion exchange chromatography and top-down mass spectrometry shows great potential.
► The method is tested with a complex mixture of 5 kDa PEG/ubiquitin conjugates.