Comparative Analysis of Different -glycan Preparation Approaches and Development of Optimized Solid-Phase Permethylation Using Mass Spectrometry.

Protein -glycosylation characterization is challenging due to structural micro- and macro-heterogeneity. Although various -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 -glycan purification strategies, including filter-aided sample preparation, de--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 -glycomics, showing simplified analysis for -glycan compositions and high yields using etanercept. The -glycan samples released from trastuzumab and adalimumab were utilized to test OSPP to obtain their -glycan profiles using mass spectrometry. Based on different standard procedures across laboratories, this study provides the reference for analysts to select an appropriate -glycan preparation method with their research purposes.