Multiplexed Antibody Glycosylation Profiling Using Dual Enzyme Digestion and Liquid Chromatography-Triple Quadrupole Mass Spectrometry Method.

Antibody glycosylation plays a crucial role in the humoral immune response by regulating effector functions and influencing the binding affinity to immune cell receptors. Previous studies have focused mainly on the immunoglobulin G (IgG) isotype owing to the analytical challenges associated with other isotypes. Thus, the development of a sensitive and accurate analytical platform is necessary to characterize antibody glycosylation across multiple isotypes. In this study, we have developed an analytical workflow using antibody-light-chain affinity beads to purify IgG, IgA, and IgM from 16 µL of human plasma. Dual enzymes, trypsin and Glu-C, were used during on-bead digestion to obtain enzymatic glycopeptides and protein-specific surrogate peptides. Ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry was used in order to determine the sensitivity and specificity. Our platform targets 95 glycopeptides across the IgG, IgA, and IgM isotypes, as well as eight surrogate peptides representing total IgG, four IgG classes, two IgA classes, and IgM. Four stable isotope-labeled internal standards were added after antibody purification to calibrate the preparation and instrumental bias during analysis. Calibration curves constructed using serially diluted plasma samples showed good curve fitting (R2 > 0.959). The intrabatch and interbatch precision for all the targets had relative standard deviation of less than 29.6%. This method was applied to 19 human plasma samples, and the glycosylation percentages were calculated, which were comparable to those reported in the literature. The developed method is sensitive and accurate for Ig glycosylation profiling. It can be used in clinical investigations, particularly for detailed humoral immune profiling.

6M50LSG Scoring System Increased the Proportion of Adequate Excess Body Weight Loss for Suspected Poor Responders After Laparoscopic Sleeve Gastrectomy in Asian Population.

PURPOSE: We aimed to evaluate the efficacy of the predictive tool, 6M50LSG scoring system, to identify suspected poor responders after laparoscopic sleeve gastrectomy (LSG). METHODS: The 6M50LSG scoring system has been applied since 2019. Suspected poor responders are defined by EBWL at 1 month < 19.5% or EBWL at 3 months < 37.7% based on the 6M50LSG scoring system. Our analysis included 109 suspected poor responders. Based on the date of LSG, the patients were separated into two groups: the 2016-2018 group (before group, BG, with regular care) and the 2019-2020 group (after group, AG, with upgrade medical nutrition therapy). RESULTS: At the end of the study, the AG group had a significantly higher proportion of adequate weight loss, which was defined as EBWL ≥ 50% at 6 months after LSG, than that in the BG group (18.92% in BG vs. 48.57% in AG, p = 0.003). The AG group demonstrated significantly more 3-months-TWL (BG: 15.22% vs. AG: 17.54%, p < 0.001) and 6-months-TWL (BG: 21.08% vs. AG: 25.65%, p < 0.001). In multivariate analyses and adjustments, the scoring system (AG) resulted in significantly higher chances of adequate weight loss in suspected poor responders (adjusted OR 3.392, 95% CI = 1.345-8.5564, p = 0.010). One year after LSG, suspected poor responders in AG had a significantly higher weight loss than those in BG (BG vs. AG: TWL 27.17% vs. 32.20%, p = 0.014) . CONCLUSION: This study confirmed that the 6M50LSG scoring system with upgraded medical nutrition therapy increased the proportion of suspected poor responders with adequate weight loss after LSG.