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Intact Glycoprotein Profiling

Glycosylation is one of the most common and important post-translational modifications (PTM) found on a protein. The alterations in glycan structures of these proteins are associated with various developmental and pathological states of glycoproteins and have great biological significance. The direct involvement of glycans in biological functions is to confer certain physiochemical properties onto proteins and to act as signals in cell-surface recognition phenomena. Glycoproteins currently constitute an active area of research. Since the functional significance of the glycan moieties have become increasingly apparent, there has been a need for rapid, reliable methods for their determination.

In the area of biotherapeutics, recombinant monoclonal antibodies (mAbs) have gained significant importance in diagnostic and therapeutic applications over the past years. The general structural features of mAbs, such as assembly of light and heavy chains via disulfide bridges, are commonly known. However, the heterogeneity of antibodies, mostly due to the variation of attached sugar moieties, requires thorough characterization. This is necessary to verify the correctness of the overall molecule and provide a reproducible, safe, and effective biological drug compound. Intact glycoprotein profiling is one such approach, in which high-resolution, accurate-mass (HR/AM) mass spectrometers are used to ascertain expected pattern and degree of glycosylation.

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Workflow Overview for Intact Glycoprotein Profiling

 
Intact glycoprotein profiling by mass spectrometry is typically done on glycoproteins that have been enriched or purified. The enriched protein mixture is introduced into the mass spectrometer using either direct infusion or liquid chromatography coupled to an ESI source. Direct infusion provides more time for signal averaging. For more complex intact-glycoprotein mixtures, on- or off-line LC separation may be required to reduce precursor spectral complexity and minimize ion suppression. Subsequent data analysis identifies the molecular weight and various glycoforms.