Rapid Evaluation of the Extent of Haptoglobin Glycosylation Using Orthogonal Intact-Mass MS Approaches and Multivariate Analysis.

Intact-mass measurements are becoming 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 is 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 the entire complement of 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) despite a significant overlap of their ionic signals. More detailed characterization of their glycosylation patterns is enabled by the recently introduced technique of cross-path reactive chromatography (XP-RC) with online MS detection, which 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. N-Glycosylation patterns of both haptoglobin phenotypes were found to be consistent with bi- and triantennary structures of complex type that exhibit significant level of fucosylation and sialylation. However, multivariate analysis of haptoglobin 1-1 reveals higher number of the triantennary structures, in comparison to haptoglobin 2-1, as well as a higher extent of fucosylation. The glycosylation patterns deduced from the XP-RC/MS measurements are in agreement with the conclusions of the intact-mass analysis supplemented by limited charge reduction, suggesting that the latter technique can be employed in situations when fast assessment of protein heterogeneity is needed (