Figure 2. Schematic flowchart of in-gel digestion of glycoproteins for N-glycan extraction.

The method is based on a protocol described in the literature [53]. Proteins are first chemically treated by reduction and alkylation. Proteins are stained with Coomassie blue and directly digested in-gel to release N-glycans. N-glycans are modified through different methods, e.g., esterification for sialic acid or labeling for reducing ends/permethylation. Quantitative analysis is carried out by fluorescence detection or mass spectrometry.