Analytical Research & Development Mass Spectrometry, MRL, Merck & Co., Inc., Kenilworth 07033, New Jersey, United States.
FyoniBio-Service Branch, Glycotope, Berlin D-13125, Germany.
Anal Chem. 2022 Jun 14;94(23):8416-8425. doi: 10.1021/acs.analchem.2c01137. Epub 2022 May 27.
CD24Fc is a homodimeric recombinant Fc-fusion protein comprised of human CD24 connected to immunoglobulin G1 (IgG1) Fc fragment. CD24 is heavily glycosylated, and its biological function is considered mainly mediated by its glycosylation. Identification of the O-glycosylation sites would facilitate an in-depth understanding of the functional role of O-glycans in CD24. However, the presence of clustered mucin-type O-glycans together with N-glycans makes the determination of O-glycosylation sites and abundance very challenging. In this study, two sets of liquid chromatography-mass spectrometry (LC-MS) workflows were developed for the comprehensive characterization of O-glycosylation in CD24: (1) Fractionation and collision-induced dissociation (CID) workflow involving multienzyme digestion, fractionation, OpeRATOR/SialEXO digestion, and CID analysis; (2) Direct OpeRATOR/SialEXO digestion followed by electron-transfer/higher-energy collision dissociation (EThcD) analysis. The precise O-glycosylation sites were identified in CD24 for the first time, and the site occupancy was assessed. A total of 12 O-glycosylation sites were identified. Seven glycosylation sites were identified by both workflows, and five additional sites were identified only by the EThcD workflow. The predominant O-glycosylation site in CD24 was Thr25 followed by Thr15. The CID workflow provided an overall relative quantitation of O-glycoforms at the CD24 level and site localization for singly O-glycosylated peptides. The EThcD workflow directly identified glycosylation sites by tandem mass spectrometry (MS/MS) for singly, doubly, and triply O-glycosylated peptides. Together, both workflows validated each other's results and can be applied to a complex mucin-type O-glycosylation site analysis of other glycoproteins and Fc-fusion therapeutics.
CD24Fc 是一种二聚体重组 Fc 融合蛋白，由与人 CD24 连接的免疫球蛋白 G1（IgG1）Fc 片段组成。CD24 高度糖基化，其生物学功能被认为主要由其糖基化介导。鉴定 O-糖基化位点将有助于深入了解 O-聚糖在 CD24 中的功能作用。然而，由于存在聚集的粘蛋白型 O-聚糖和 N-聚糖，因此确定 O-糖基化位点和丰度极具挑战性。在这项研究中，开发了两套用于全面表征 CD24 中 O-糖基化的液相色谱-质谱（LC-MS）工作流程：（1） 分步和碰撞诱导解离（CID）工作流程，涉及多酶消化、分步、OpeRATOR/SialEXO 消化和 CID 分析；（2） 直接 OpeRATOR/SialEXO 消化，然后进行电子转移/更高能量碰撞解离（EThcD）分析。首次在 CD24 中鉴定出精确的 O-糖基化位点，并评估了其占有率。总共鉴定出 12 个 O-糖基化位点。两种工作流程均鉴定出 7 个糖基化位点，而 EThcD 工作流程仅鉴定出另外 5 个位点。CD24 中主要的 O-糖基化位点是 Thr25，其次是 Thr15。CID 工作流程提供了 CD24 水平上 O-糖型的总体相对定量和单糖基化肽的位点定位。EThcD 工作流程通过串联质谱（MS/MS）直接鉴定单糖基化、双糖基化和三糖基化肽的糖基化位点。总之，两种工作流程相互验证，可应用于其他糖蛋白和 Fc 融合治疗药物的复杂粘蛋白型 O-糖基化位点分析。
