Glycoprotein Enrichment and N-Glycosylation Analysis

Based on specific capture of glycoproteins by hydrazide resin, the N-glycosylation sites were analyzed and identified using the LC–MS/MS (Thermo Scientific, MA, USA) (Zhang et al. 2003 (link)) (Fig. 1). 1 mg EcXly were dissolved in coupling buffer (100 mM NaAc, 150 mM NaCl), and 15 mM NaIO₄ was added to oxidize the proteins for 1 h. NaIO₄ was then removed using the ultrafiltration tube, and the proteins were coupled with hydrazide resin (Bio-Rad, USA) at room temperature for 10–24 h. To remove the nonglycoproteins, the sample was washed six times using an equal volume of buffer. The proteins were reduced by adding 20 mM dithiothreitol at 60 °C for 1 h and subsequently alkylated by the addition of 20 mM iodoacetamide in the dark for 40 min. The trypsin was used to hydrolyze proteins into peptides, and the PNGase F was used to release the enriched glycopeptides from hydrazide resin. Finally, the released peptides were desalinated and resuspended in an appropriate amount of 0.1% formic acid for further LC–MS/MS analysis. The peptides of MiXly and the mutants of EcXly were obtained by in-gel digestion and used for LC–MS/MS analysis (De Godoy et al 2006 (link)). The acquired MS data were analyzed using Proteome Discoverer 2.2.1 software as previously described (Yuan et al. 2022 (link)).Fig. 1

The schematic workflow of glycosylation analysis using LC–MS/MS