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  • P-ISSN2233-4203
  • E-ISSN2093-8950

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

Mass Spectrometry Letters, (P)2233-4203; (E)2093-8950
2021, v.12 no.3, pp.66-75
https://doi.org/10.5478/10.5478/MSL.2021.12.3.66
Ju Yeon Lee (Korea Basic Science Institute)
Jin-woong Choi (Korea Basic Science Institute)
Sanghyeon Bae (Korea Basic Science Institute)
Heeyoun Hwang (Korea Basic Science Institute)
Jong Shin Yoo (Korea Basic Science Institute)
Joo Eon Lee (Osong Medical Innovation Foundation)
Eunji Kim (Azcuris, Co., Ltd.)
Young Ho Jeon (Azcuris, Co., Ltd.)
Jin Young Kim (Korea Basic Science Institute)

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Abstract

Interleukin-4 (IL-4) and IL-13 are cytokines secreted by immune cells. Cytokines induce the proliferation of macro-phages 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 chro-matography–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-glycopep-tides, 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%, respec-tively), and the biantennary glycans were the most abundant in both recombinant IL-4 and IL-13 proteins.

Submission Date
2021-08-17
Revised Date
2021-09-07
Accepted Date
2021-09-14

Mass Spectrometry Letters