Proteoextract glycopeptide enrichment kit

Purified IgGs were digested by trypsin and glycopeptides were isolated from peptides using two methods, i.e., reversed-phase high-performance liquid chromatography and a protocol involving the commercial ProteoExtract^® Glycopeptide Enrichment Kit (EMD-Millipore, Etobicoke, ON, Canada). Fractions were concentrated for analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS). The instrument used was an UltraFleXtreme™ (Bruker, Bremen, Germany) operated in positive ion, reflective mode.

Aliquots of 150 μg to 200 μg of the VLPs were proteolytically digested using trypsin, chymotrypsin, and GluC (Promega). ProteoExtract Glycopeptide Enrichment Kit (Merck Millipore) was used to enrich glycopeptides for LC-ESI MS. For site-specific analysis of LASV GPC glycosylation, enriched glycopeptides were analyzed on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) coupled to an EASY-Spray nano-LC system (Thermo Fisher Scientific). MS data were acquired with XCalibur 4.0 (Thermo Fisher Scientific). Glycopeptides were fragmented using both higher-energy collisional dissociation and energy transfer dissociation. Data analysis and glycopeptide identification were performed using Byonic and Byologic software (Protein Metrics).

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)).

A 200- to 300-μg sample of trimer was used for in-solution proteolytic digestion using trypsin or chymotrypsin (Mass Spectrometry Grade, Promega), followed by enrichment of the digestion mixture for glycopeptides using the ProteoExtract Glycopeptide Enrichment Kit (Merck Millipore). Glycopeptides were then either directly analyzed by LC-ESI MS or fractionated by RP-HPLC and subjected to MALDI-TOF MS.

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)).