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 proteo forms 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 electro sprayionization (ESI) MS is an elegant way to obtain meaningfulinformation on most heterogeneous systems, yielding not only theaverage 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 ionicsignals. 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 ofdisulfide bonds to generate metastable haptoglobin subunits. Application of XP-RC to both haptoglobin phenotypes confirms thatno modifications are present within their light chains and provides a wealth of information on glycosylation patterns of the heavychains. N-Glycosylation patterns of both haptoglobin phenotypes were found to be consistent with bi- and triantennary structures ofcomplex type that exhibit significant level of fucosylation and sialylation. However, multivariate analysis of haptoglobin 1-1 revealshigher number of the triantennary structures, in comparison to haptoglobin 2-1, as well as a higher extent of fucosylation. Theglycosylation 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 (e.g., process analytical technology applications).

在基于质谱（MS）的蛋白质表征中，完整质量测量正变得越来越流行，因为它们能够在相对较短的时间内对蛋白质的所有变体形式进行评估。然而，这种方法目前的应用仅限于结构多样性相对适中的系统，因为高度的异质性常常妨碍直接进行质谱测量。在电喷雾电离（ESI）质谱中引入有限的电荷减少是一种获取大多数异质系统有意义信息的巧妙方法，它不仅能得出蛋白质的平均质量，还能得出蛋白质所有变体形式所涵盖的质量范围。将这种方法应用于触珠蛋白两种不同表型（1 - 1和2 - 1）的表征，提供了证据表明尽管它们的离子信号有明显重叠，但它们的糖基化程度存在显著差异（表型2 - 1的聚糖负载明显较轻）。最近引入的具有在线质谱检测的交叉路径反应色谱（XP - RC）技术能够对它们的糖基化模式进行更详细的表征，该技术将色谱分离与在线二硫键还原相结合，以产生亚稳态的触珠蛋白亚基。将XP - RC应用于两种触珠蛋白表型证实了它们的轻链内没有修饰，并提供了大量关于重链糖基化模式的信息。发现两种触珠蛋白表型的N - 糖基化模式与复合型的双天线和三天线结构一致，这些结构表现出显著的岩藻糖基化和唾液酸基化水平。然而，对触珠蛋白1 - 1的多变量分析显示，与触珠蛋白2 - 1相比，其三天线结构数量更多，岩藻糖基化程度也更高。从XP - RC/质谱测量中推断出的糖基化模式与通过有限电荷减少补充的完整质量分析的结论一致，这表明后一种技术可用于需要快速评估蛋白质异质性的情况（例如，过程分析技术应用）。