Analysis of rhIL-12 Disulfide Bond And N-glycosylation Sites and C-terminal Amino Acid Sequence

doi:10.13523/j.cb.20140506

China Biotechnology

2014, Vol. 34

Issue (5): 39-53 DOI: 10.13523/j.cb.20140506

Analysis of rhIL-12 Disulfide Bond And N-glycosylation Sites and C-terminal Amino Acid Sequence

ZHAO Feng¹, ZHANG Yi-jun², RAN Yan-hong¹, WANG Xing-yong², YE Qian-jun², LI Hong-jian¹

1 College of Life Science and Technology, Jinan University, Guangzhou 510632, China;
2 Guangzhou Kai Tai Biological Engineering Co., LTD Guangzhou 510663, China

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Abstract

Recombinant human interleukin- 12 (rhIL-12) is a heterodimeric glycoprotein that has been used to treat diseases such as tumor, parasites, viral infections and hematopoietic disorders. For the structure confirmation is important for quality control, in this paper, the disulfide linkage, N-glycosylation sites and C-terminal amino acid sequences of rhIL-12 expressed by CHO cells were analyzed, and the rhIL-12 underwent enzymolysis using three non-reducing enzymes: Trypsin, Chymotrypsin and Glu-C, to break between cysteine residues and form the disulfide-linked peptides, and then peptide samples were analyzed using LC-MS/MS to identify seven pairs of disulfide bonds presenting in rhIL-12 sample that match the theoretical conditions. After reduction of disulfide bond and alkylation modification protection, the rhIL-12 underwent enzymolysis using Trypsin, Chymotrypsin and GluC, and mass spectrum peptide mapping and C-terminal amino acid sequence analysis of the peptide segments were then carried out using LC-MS/MS, to identify eight amino acids at p35 subunit C-terminal and 15 amino acids at p40subunit C-terminal of rhIL-12. After reduction and alkylation, the rhIL-12 samples were degenerated and enzymolysised, and the enzyme-digested products of peptide segments were thereafter treated with PNGase F in H₂O and H₂¹⁸O respectively. Through tandem mass spectrometry analysis of the change in molecular weight of peptide fragments, three N glycosylation sites of rhIL-12 were exactly identified, which were site 71 and site85 of p35 subunit, and site 200 of p40 subunit. Through establishing the method combining enzymolysis with mass spectrometry identification, ultimately it was demonstrated that disulfide bond site, C-terminal amino acid sequence and glycosylation sites of new drug rhIL-12 were consistent with the theoretical conditions.

Key words： rhIL-12 disulfide bone N-glycosylation sites C-terminal amino acid sequence LC-MS/MS

Received: 11 March 2014 Published: 25 May 2014

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Cite this article:

ZHAO Feng, ZHANG Yi-jun, RAN Yan-hong, WANG Xing-yong, YE Qian-jun, LI Hong-jian. Analysis of rhIL-12 Disulfide Bond And N-glycosylation Sites and C-terminal Amino Acid Sequence. China Biotechnology, 2014, 34(5): 39-53.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20140506 OR https://manu60.magtech.com.cn/biotech/Y2014/V34/I5/39

[1] Christina Yoon, Steven C. Johnston, et al. Charged residues dominate a unique interlocking topography in the heterodimeric cytokine interleukin-12. The EMBO Journal, 2000,19(14): 3530-3541.

[2] Del Vecchio M, Bajetta E, Canova S,et al. Interleukin-12: biological properties and clinical application. Clin Cancer Res,2007,13(16):4677-4685.

[3] Majumdar M K,Thiede M A,Mosca J D,el al.Phenotypic and functional comparison of cultures of marrow derived mesenchymal stem cells(MSCs)and stromal cells.J Cell Physiol,I998,176:57-66.

[4] Tingchao Chen.IL-12 Facilitates both the recovery of endogenous hematopoiesis and the engraftment of stem cells after ionizing radiation.Experimental Hematology, 2007,35: 203-213.

[5] Lena A Basile. Multilineage hematopoietic recovery with concomitant antitumor effects using low dose Interleukin-12 in myelosuppressed tumor-bearing mice. Journal of Translational Medicine, 2008: 6-26.

[6] Gonzalez J,Takao T,et al. A method for determination of N-glycosylation sites in glycoproteins by collision-induced dissociation analysis in fast atom bombardment mass spectrometry:identification of the positions of carbohydrate linked asparagine in recombinant alpha-amylase by treatment with peptide-N-glycosidase F in ¹⁸O labeled water. Anal.Biochem,1992,205 (1):151-158.

[7] Kuster B, Mann M.¹⁸O-labeling of N-glycosylation sites to improve the identification of gel-separated glycoproteins using peptide mass mapping and database searching. Anal.Chem,1999,71 (7):1431-1440.

[8] Kristiansen T Z, Bunkenborg J, et al. A proteomic analysis of human bile. Mol.Cell.Proteomics, 2004,3 (7):715-728.

[9] Kaji H,Saito H,et al.Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins.Nat.Biotechnol,2003,21 (6): 667 -672.

[10] Per Hagglund,Rune Matthiesen,et al.An enzymatic deglycosylation scheme enabling identification of core fucosylated N-Glycans and O-Glycosylation Site Mapping of Human Plasma Proteins.Journal of Proteome Research,2007,6:3021-3031.

[11] Anthony R,Mire-Sluis,Robin T.Cytokine,london:Academic press,1998.

[12] Hulsmeier A J, Paesold-Burda P, Hennet T. N-glycosylation site occupancy in serum glycoproteins using multiple reaction monitoring liquid chromatography-mass spectrometry. Mol.Cell.Proteomics, 2007(6): 2132-2138.

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