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http://dx.doi.org/10.5478/MSL.2021.12.3.66

Characterization of the N-glycosylation of Recombinant IL-4 and IL-13 Proteins Using LC-MS/MS Analysis and the I-GPA Platform  

Lee, Ju Yeon (Research Center for Bioconvergence Analysis, Korea Basic Science Institute)
Choi, Jin-woong (Research Center for Bioconvergence Analysis, Korea Basic Science Institute)
Bae, Sanghyeon (Research Center for Bioconvergence Analysis, Korea Basic Science Institute)
Hwang, Heeyoun (Research Center for Bioconvergence Analysis, Korea Basic Science Institute)
Yoo, Jong Shin (Research Center for Bioconvergence Analysis, Korea Basic Science Institute)
Lee, Joo Eon (New Drug Development Center, Osong Medical Innovation Foundation)
Kim, Eunji (Azcuris, Co., Ltd.)
Jeon, Young Ho (Azcuris, Co., Ltd.)
Kim, Jin Young (Research Center for Bioconvergence Analysis, Korea Basic Science Institute)
Publication Information
Mass Spectrometry Letters / v.12, no.3, 2021 , pp. 66-75 More about this Journal
Abstract
Interleukin-4 (IL-4) and IL-13 are cytokines secreted by immune cells. Cytokines induce the proliferation of macrophages or promote the differentiation of secretory cells. The initiation and progression of allergic inflammatory diseases, such as asthma, are dependent on cytokines acting through related receptor complexes. IL-4 and IL-13 are N-glycoproteins. Glycan structures in glycoproteins play important roles in protein folding, protein stability, enzymatic function, inflammation, and cancer development. Therefore, the glycan structure of IL-4 and IL-13 needs to be elucidated in detail for the development of effective therapies. We report the first attempt to characterize the site-specific N-glycosylation of recombinant IL-4 and IL-13 via liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The tandem mass spectra of intact N-glycopeptides were identified using the Integrated GlycoProteome Analyzer (I-GPA) platform, which can automatically and rapidly analyze multiple N-glycopeptides, including their glycan composition and amino acid sequences. The recombinant IL-4 and IL-13 were identified with amino acid sequence coverages of 84% and 96%, respectively. For IL-4, 52 glycoforms on one N-glycosylation site were identified and quantified. In IL-13, 232 N-glycopeptides from three N-glycosylation sites were characterized, with the site Asn52 being the most extensively glycosylated (~80%). The complex glycans were the most abundant glycan on IL-4 and IL-13 (~96% and 91%, respectively), and the biantennary glycans were the most abundant in both recombinant IL-4 and IL-13 proteins.
Keywords
site-specific N-glycosylation; LC-MS/MS; I-GPA;
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