Sialylated N-glycan isomers with a2-3 or a2-6 linkage(s) have distinctive roles in glycoproteins, but are difficult to distinguish. Wild-type (WT) and glycoengineered (mutant) therapeutic glycoproteins, cyto-toxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig), were produced in Chinese ham-ster ovary cell lines; however, their linkage isomers have not been reported. In this study, N-glycans of CTLA4-Igs were released, labeled with procainamide, and analyzed by liquid chromatography-tandem mass spectrometry (MS/MS) to identify and quantify sialylated N-glycan linkage isomers. The linkage isomers were distinguished by comparison of 1) intensity of the N-acetylglucosamine ion to the sialic acid ion (Ln/Nn) using different fragmentation stability in MS/MS spectra and 2) retention time-shift for a selective m/z value in the extracted ion chromatogram. Each isomer was distinctively identified, and each quantity (>0.1%) was obtained relative to the total N-glycans (100%) for all observed ionization states. Twenty sialylated N-glycan isomers with only a2-3 linkage(s) in WT were identified, and each isomer's sum of quantities was 50.4%. Furthermore, 39 sialylated N-glycan isomers (58.8%) in mono-(3 N-glycans; 0.9%), bi-(18; 48.3%), tri-(14; 8.9%), and tetra-(4; 0.7%) antennary structures of mutant were obtained, which comprised mono-(15 N-glycans; 25.4%), di-(15; 28.4%), tri-(8; 4.8%), and tetra-(1; 0.2%) sialy-lation, respectively, with only a2-3 (10 N-glycans; 4.8%), both a2-3 and a2-6 (14; 18.4%), and only a2-6 (15; 35.6%) linkage(s). These results are consistent with those for a2-3 neuraminidase-treated N-glycans. This study generated a novel plot of Ln/Nn versus retention time to distinguish sialylated N-glycan linkage isomers in glycoprotein.(c) 2023 The Author(s). Published by Elsevier B.V. on behalf of Xi'an Jiaotong University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
