Chymotrypsin mass spectrometry grade

Site-specific N-glycosylation analysis was performed using proteolytic digestion followed by tandem LC-MS. Prior to digestion, trimers were denatured, reduced and alkylated by incubation for 1h at room temperature (RT) in a 50 mM Tris/HCl, pH 8.0 buffer containing 6 M urea and 5 mM dithiothreitol (DTT), followed by the addition of 20 mM iodacetamide (IAA) for a further 1h at RT in the dark, and then additional DTT (20 mM) for another 1h, to eliminate any residual IAA. The alkylated trimers were buffer-exchanged into 50 mM Tris/HCl, pH 8.0 using Vivaspin columns (GE healthcare) and digested separately with trypsin, elastase and chymotrypsin (Mass Spectrometry Grade, Promega) at a ratio of 1:30 (w/w). Glycopeptides were selected from the protease-digested samples using the ProteoExtract Glycopeptide Enrichment Kit (Merck Millipore) following the manufacturer’s protocol. Enriched glycopeptides were analyzed by LC-ESI MS on an Orbitrap fusion mass spectrometer (Thermo Fisher Scientific), as previously described (Behrens et al., 2016 (link)), using higher energy collisional dissociation (HCD) fragmentation. Data analysis and glycopeptide identification were performed using ByonicTM (Version 2.7) and ByologicTM software (Version 2.3; Protein Metrics Inc.), as previously described (Behrens et al., 2016 (link)).

BG505 SOSIP proteins (100-150 μg each) were buffer exchanged using Vivaspin 100 kDa columns, denatured, reduced, and alkylated by sequential 1 h incubations at room temperature (RT) in the following solutions: 50 mM Tris/HCl, pH 8.0 buffer containing 6 M urea and 5 mM dithiothreitol (DTT), followed by the addition of 20 mM iodacetamide (IAA) for a further 1h at RT in the dark, and then additional DTT (20 mM), to eliminate residual IAA. The proteins were then buffer-exchanged into 50 mM Tris/HCl, pH 8.0 using Vivaspin 3 kDa columns and aliquots were digested with trypsin or chymotrypsin (Mass Spectrometry Grade, Promega) at a ratio of 1:30 (w/w) for 16 h at 37°C. The reactions were dried and glycopeptides were extracted using C18 Zip-tip (Merck Millipore) following the manufacturer’s protocol. Briefly, tips were equilibrated by alternating in acetonitrile and 0.1% trifluoracetic acid. The reaction mixture was loaded on to the tip and eluted with 50% acetonitrile, 0.1% trifluoracetic acid.