Enriching Glycopeptides for Mass Spectrometry

Before proteolytic digestion, trimers were denatured and alkylated by incubation for 1 hr 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 1 hr 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 and digested with trypsin and elastase (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). Enriched glycopeptides were analyzed by LC-ESI MS on an Orbitrap fusion mass spectrometer (ThermoFisher Scientific), as previously described (Behrens et al., 2016 (link)), using higher energy collisional dissociation (HCD) fragmentation. Data analysis and glycopeptide identification were performed using Byonic™ (Version 2.7) and Byologic™ software (Version 2.3; Protein Metrics Inc.), as previously described (Behrens et al., 2016 (link)).