Abstract Heparin is a sulfated glycosaminoglycan, and the most used pharmaceutical anticoagulant worldwide. Heparin is isolated primarily from porcine sources, and possesses a complex variable structure, wherein anticoagulant activity is principally derived from the pentasaccharide GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA) motif, containing an infrequent 3-O-sulfated glucosamine that enhances antithrombin stabilization. Pedobacter heparinus heparin lyases are currently widely employed for characterization of heparin and its derivatives, but have limited activity near regions of 3-O-sulfation, hindering analyses of anticoagulation relevant sequences. Here, using a panel of biophysical and structural techniques, we show that Bacteroides eggerthii lyase I liberates ΔUA-GlcNS3S6S disaccharides from the defined pentasaccharide, fondaparinux. This activity provides improved access to 3-O-sulfated heparin standards, with potential to aid heparin quality control.
Abstract IgG is the most common scaffold used for antibody-based therapeutics. Utilization of the ever-expanding knowledge base of the IgG scaffold provides a wealth of design options for therapeutic development. These therapeutics apply varied engineering strategies in various combinations utilizing three key IgG features: the crystallizable fragment (Fc), antigen-binding fragment (Fab), and the conserved N-glycans. Designs may be as simple as amino acid variants to modulate effector function or as complex as large designs incorporating additional Fab, Fc, and non-IgG domain fusions to modulate effector molecule recognition, valency, and antigen specificity. The engineering strategies and basic knowledge supporting these developments have been the subject of rapid expansion. Here we review multiple approaches and advances with a focus on the last five years.
Abstract N-glycolylneuraminic acid (Neu5Gc) has been repeatedly associated with human cancer, but reliable detection in mass-spectrometric studies has remained elusive, but its low abundance has led to inconsistent detection in studies of human samples. To address this, matrix-matched calibration curves, which have been pioneered in proteomics and metabolomics for assessing changes in complex mixtures, were measured of released N-glycans at four orders of magnitude dynamic range in defined mixtures, systematically benchmarking Neu5Gc-containing N-glycan detection across multiple LC–MS platforms and sites. Orthogonally, the gold-standard analytical method, consisting of fluorescence detection of labelled monosaccharides separated by LC, was applied to the same samples, yielding absolute concentrations of Neu5Gc. LC–MS demonstrated an extended detection range of three or more orders of magnitude while retaining intact N-glycan measurement, improving assay specificity and enabling detection of the variety of Neu5Gc-bearing N-glycans. By combining orthogonal dimensions of evidence, including chromatographic separation, isotopic distribution matching, and composition-confirming MS/MS, LC–MS confidently resolved Neu5Gc signals from noise, even at low abundance. In comparison, under the DMB-LC-FLR conditions and sample loading evaluated here, dynamic range was limited to two orders of magnitude, insufficient for detection of Neu5Gc in commercially available pooled human sera. These findings strongly support that the DMB-LC-FLR assay’s specificity and sensitivity were insufficient for Neu5Gc detection in human samples due to noise overwhelming the Neu5Gc signal. By establishing a reusable benchmarking framework for future glycomic studies, we aim to use LC–MS to improve the measurement of Neu5Gc in clinical samples.
