Comparative Analysis of Different N-glycan Preparation Approaches and Development of Optimized Solid-Phase Permethylation Using Mass Spectrometry
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- Erscheinungsjahr:
- 2021
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Protein N-glycosylation characterization is challenging due to structural micro- and macro-heterogeneity. Although various N-glycan preparation strategies, including purification and derivatization, have been previously developed prior to mass spectrometric analysis, systematic evaluation still needs to be performed. This study compared the different N-glycan purification strategies, including filter-aided sample preparation, de-N-glycosylated protein precipitation, and trypsin digestion followed by reversed phase-based solid-phase extraction, and derivatization approaches, such as solid-phase permethylation, reductive amination, and reduction. With the comparative analysis, an optimized solid-phase permethylation (OSPP) workflow was developed for mass spectrometric N-glycomics, showing simplified analysis for N-glycan compositions and high yields using etanercept. The N-glycan samples released from trastuzumab and adalimumab were utilized to test OSPP to obtain their N-glycan profiles using mass spectrometry. Based on different standard procedures across laboratories, this study provides the reference for analysts to select an appropriate N-glycan preparation method with their research purposes.
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- info:eu-repo/semantics/closedAccess
- Quellsystem:
- Forschungsinformationssystem des UKE
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- oai:pure.atira.dk:publications/d55e2b6b-88f1-4886-bae7-3455d75675c3