An integrated approach to analyze EG2-hFc monoclonal antibody N-glycosylation by MALDI-MS
| العنوان: | An integrated approach to analyze EG2-hFc monoclonal antibody N-glycosylation by MALDI-MS |
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| المؤلفون: | Emy Komatsu, Edward Bodnar, Michael Butler, Maureen Spearman, Paul Lopez, Yann Zogbi, Andrey Giovanni Gomes de Oliveira, Venkata S. Tayi, Tristan Alexander Smythe, Thais Ferreira Nascimento, Rini Roy, Hélène Perreault, Lauren Girard |
| المصدر: | Canadian Journal of Chemistry. 93:754-763 |
| بيانات النشر: | Canadian Science Publishing, 2015. |
| سنة النشر: | 2015 |
| مصطلحات موضوعية: | Gel electrophoresis, PNGase F, Glycan, Chromatography, biology, Chemistry, medicine.drug_class, Chinese hamster ovary cell, Organic Chemistry, General Chemistry, Monoclonal antibody, Catalysis, Glycopeptide, N-linked glycosylation, Exoglycosidase, biology.protein, medicine |
| الوصف: | The characterization of the N-glycan portion of antibodies has been the subject of several studies involving mass spectrometry. In this article, a workflow is presented that starts with the expression of a monoclonal antibody (EG2-hFc) in Chinese hamster ovary cells and continues with Protein A purification of the antibody. Then the protocol continues with gel electrophoresis. Bands containing the heavy chain are cut and isolated from the gel followed by tryptic digestion to obtain peptides and glycopeptides. The enrichment of glycopeptides by C18 chromatography is described followed by characterization using positive and negative modes MALDI-MS and MS/MS. An exoglycosidase, beta-galactosidase, is used to verify anomericity of linkages in the glycan portion of glycopeptides. In the last step, glycans are detached from glycopeptides using PNGase F labelled with phehylhydrazine and characterized by MALDI-MS/MS. This workflow is reported for the first time for this particular antibody and presents a valuable approach for the analysis of N-glycans on most antibodies and glycoproteins. |
| تدمد: | 1480-3291 0008-4042 |
| DOI: | 10.1139/cjc-2015-0061 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_________::7cde170b333bd0972f1df6d332c2d484 https://doi.org/10.1139/cjc-2015-0061 |
| Rights: | CLOSED |
| رقم الانضمام: | edsair.doi...........7cde170b333bd0972f1df6d332c2d484 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 14803291 00084042 |
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| DOI: | 10.1139/cjc-2015-0061 |