Towards middle-up analysis of polyclonal antibodies: subclass-specific N-glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC-MS
| Autor: | Therese Wohlschlager, Petra Winter, Christof Regl, Richard Weiss, Christian G. Huber, Constantin Blöchl |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Proteomics
0301 basic medicine Glycosylation medicine.drug_class Protein subunit Lysine Glycobiology lcsh:Medicine Monoclonal antibody 01 natural sciences Antibodies Article Subclass Immunoglobulin G Mice 03 medical and health sciences N-linked glycosylation medicine Animals lcsh:Science Chromatography High Pressure Liquid Glycoproteins Mice Inbred BALB C Multidisciplinary Mass spectrometry biology Chemistry Immunochemistry lcsh:R Vaccination 010401 analytical chemistry Bioanalytical chemistry Allergens Antigens Plant Molecular biology Immunoglobulin Fc Fragments 0104 chemical sciences Mice Inbred C57BL 030104 developmental biology Polyclonal antibodies biology.protein lcsh:Q Female Antibody Protein Processing Post-Translational Analytical chemistry |
| Zdroj: | Scientific Reports Scientific Reports, Vol 10, Iss 1, Pp 1-12 (2020) |
| ISSN: | 2045-2322 |
| Popis: | In recent years, advanced HPLC-MS strategies based on intact protein (“top-down”) or protein subunit (“middle-up/middle-down”) analysis have been implemented for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up/middle-down analysis for polyclonal IgGs exhibiting extensive sequence variability. Specifically, we addressed IgGs from mouse, representing an important model system in immunological investigations. To obtain Fc/2 portions as conserved subunits of IgGs, we made use of the bacterial protease SpeB. For this purpose, we initially determined SpeB cleavage sites in murine IgGs. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry. This enabled simultaneous relative quantification of IgG subclasses and their N-glycosylation variants, both of which influence IgG effector functions. To assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. The study revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts, respectively. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine, and may thus be implemented for quality control of functional antibodies. |
| Databáze: | OpenAIRE |
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