Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids
| Autor: | Ian C. Schoenhofen, Yves Durocher, Paul Lopez, Michael Butler, Edward Bodnar, Céline Raymond, Hélène Perreault, Carina Villacrés |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Glycan
CHO Cells Sialylation Biochemistry MALDI-ToF-MS Analytical Chemistry Metabolic engineering chemistry.chemical_compound Cricetulus Polysaccharides Cricetinae N-Acetyllactosamine Synthase Animals Humans Molecular Structure biology Chemistry Chinese hamster ovary cell Enzymatic sialylation Glycome Sialic acid Azido-sialic acid Matrix-assisted laser desorption/ionization Metabolic sialylation Metabolic Engineering Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Sialic Acids biology.protein Click chemistry Monoclonal antibodies Bioorthogonal chemistry |
| Zdroj: | Analytical and Bioanalytical Chemistry. 407:8945-8958 |
| ISSN: | 1618-2650 1618-2642 |
| DOI: | 10.1007/s00216-015-9010-x |
| Popis: | Metabolic engineering of glycans present on antibodies and other glycoproteins is becoming an interesting research area for improving our understanding of the glycome. With knowledge of the sialic acid biosynthetic pathways, the experiments described in this report are based on a published procedure involving the addition of a synthesized azido-mannosamine sugar into cell culture media and evaluation of downstream expression as azido-sialic acid. This unique bioorthogonal sugar has the potential for a variety of “click chemistry” reactions through the azide linkage, which allow for it to be isolated and quantified given the choice of label. In this report, mass spectrometry was used to investigate and optimize the cellular absorption of peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to form N-azidoacetylneuraminic acid (SiaNAz) in a Chinese hamster ovary (CHO) cell line transiently expressing a double mutant trastuzumab (TZMm2), human galactosyltransferase 1 (GT), and human α-2,6-sialyltransferase (ST6). This in vivo approach is compared to in vitro enzymatic addition SiaNAz onto TZMm2 using soluble β-galactosamide α-2,6-sialyltransferase 1 and CMP-SiaNAz as donor. The in vivo results suggest that for this mAb, concentrations above 100 μM of Ac4ManNAz are necessary to allow for observation of terminal SiaNAz on tryptic peptides of TZMm2 by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This is further confirmed by a parallel study on the production of EG2-hFc monoclonal antibody (Zhang J et al. Prot Expr Purific 65(1); 77–82, 2009) in the presence of increasing concentrations of Ac4ManNAz. |
| Databáze: | OpenAIRE |
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