Evaluation of the Extent of Haptoglobin Glycosylation Using Orthogonal Intact-Mass MS Approaches

05 January 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Intact-mass measurements are becoming an increasingly popular in mass spectrometry (MS) based protein characterization, as they allow the entire complement of proteoforms to be evaluated within a relatively short time. However, applications of this approach are currently limited to systems exhibiting relatively modest degrees of structural diversity, as the high extent of heterogeneity frequently prevents straightforward MS measurements. Incorporation of limited charge reduction into electrospray ionization (ESI) MS measurements provides an elegant way to obtain meaningful information on most heterogeneous systems, yielding not only the average mass of the protein, but also the mass range populated by various proteoforms. Application of this approach to characterization of two different phenotypes of haptoglobin (1-1 and 2-1) provides evidence of a significant difference in their extent of glycosylation, with the glycan load of phenotype 2-1 being notably lighter. More detailed characterization of their glycosylation patterns is enabled by the recently introduced crosspath reactive chromatography (XP-RC) with on-line MS detection, a technique that combines chromatographic separation with in-line reduction of disulfide bonds to generate metastable haptoglobin subunits. Application of XP-RC to both haptoglobin phenotypes confirms that no modifications are present within their light chains, and provides a wealth of information on glycosylation patterns of the heavy chains. The haptoglobin 1-1 glycans are mature fully sialylated biantennary structures that exhibit high degrees of fucosylation. In contrast, phenotype 2-1 contains a significant fraction of incomplete biantennary structures and exhibit significantly lower levels of sialylation and fucosylation. The glycosylation patterns deduced from the XP-RC/MS measurements are in agreement with the conclusions of haptoglobin analysis by limited charge reduction, suggesting that the latter can be employed in situations when a fast assessment of a protein heterogeneity is needed (e.g., comparability studies of biopharmaceutical products).


ion chemistry
disulfide reduction


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