Modern analytical
approaches employing high-resolution mass spectrometry
(MS) facilitate the generation of a vast amount of structural data
of highly complex glycoproteins. Nevertheless, systematic interpretation
of this data at different structural levels remains an analytical
challenge. The glycoprotein utilized as a model system in this study,
human chorionic gonadotropin (hCG), exists as a heterodimer composed
of two heavily glycosylated subunits. In order to unravel the multitude
of glycoforms of recombinant hCG (drug product Ovitrelle), we combine
established techniques, such as released glycan and glycopeptide analysis,
with novel approaches employing high-performance liquid chromatography-mass
spectrometry (HPLC-MS) to characterize protein subunits and native
MS to analyze the noncovalent hCG complex. Starting from the deconvoluted
mass spectrum of dimeric hCG comprising about 50 signals, it was possible
to explore the chemical space of hCG glycoforms and elucidate the
complexity that hides behind just 50 signals. Systematic, stepwise
integration of data obtained at the levels of released glycans, glycopeptides,
and subunits using a computational annotation tool allowed us to reveal
1031 underlying glycoforms. Additionally, critical quality attributes
such as sialylation and core fucosylation were compared for two batches
of Ovitrelle to assess the potential product variability.
Exploration of a highly glycosylated non-covalent protein comples by mass spectreometry is a challenging task due to isobarisicy of the majority of glycoforms. Integration of data from multiple structural levels (released glycan-glycopeptide-protein subunit-protein complex) by means of computational algorithms permits unraveling the hidden diversity of the human chorionic gonadotropin heterodimer, constituting the base for the study of complex glycosylated protein assemblies.<br>
Exploration of a highly glycosylated non-covalent protein comples by mass spectreometry is a challenging task due to isobarisicy of the majority of glycoforms. Integration of data from multiple structural levels (released glycan-glycopeptide-protein subunit-protein complex) by means of computational algorithms permits unraveling the hidden diversity of the human chorionic gonadotropin heterodimer, constituting the base for the study of complex glycosylated protein assemblies.<br>
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