Journal: Methods in enzymology

Article Title: Chemoenzymatic glycan remodeling of natural and recombinant glycoproteins

doi: 10.1016/bs.mie.2017.06.006

Figure Lengend Snippet: Mass spectrometry analysis of glycan remodeling of EPO. A) MALDI-TOF analysis of glycan released from EPO with high-mannose type glycan. The glycan release and MALDI-TOF analysis was performed as described in (Yang & Wang, 2016). B) LC-MS analysis of GlcNAc-EPO (EPO-Gn). C) LC-MS analysis of EPO transferred with M3N3 glycan.

Article Snippet: 2.1 Glycoproteins, enzymes, and substrates Monoclonal antibody Herceptin: Genentech Inc. (San Francisco, CA) GlcNAc-Herceptin Fucα1,6-GlcNAc-Herceptin EPO (His-tagged) with high-mannose type glycan ( Yang & Wang, 2016 ) GlcNAc-EPO Endo Hf: New England Biolabs (Ipswich, MA) Endo-A ( Umekawa et al., 2008 ) Endo-S2 ( Li et al., 2016 ) EndoS2-D184M ( Li et al., 2016 ) AlfC (α1,6-Fucosidase) ( Rodriguez-Diaz et al., 2011 ) PNGase F: New England Biolabs (Ipswich, MA) Sial 2 Gal 2 GlcNAc 2 Man 3 GlcNAc oxazoline (S2G2-Oxa) ( Seko et al., 1997 ) Man 9 GlcNAc 2 oxazoline (M9-Oxa) ( Wang et al., 2004 ) Azide-tagged Man 3 GlcNAc oxazoline (M3 N3 -Oxa) ( Ochiai et al., 2008 ) 2.2 Buffers, columns, and reagents HiTrap Protein A column (GE Healthcare, Little Chalfont, UK) HisTrap Ni-NTA column (GE Healthcare, Little Chalfont, UK) Poroshell 300SB-C8 column 5 μm 1 × 75 mm (Agilent, Santa Clara, CA) XBridge BEH300 C4 column 3.5 μm 2.1 × 50 mm (Waters, Milford, MA) Amicon Ultra Centrifugal Filters Ultracel 30,000 NMWL (EMD Millipore, Billerica, MA) LC-MS buffer A: ddH 2 O with 0.1% formic acid LC-MS buffer B: acetonitrile with 0.1% formic acid Protein A wash buffer: Phosphate-buffered saline (PBS), pH 7.4 Protein A elution buffer: 100 mM Glycine-HCl, pH 2.7 Protein A neutralization buffer: 1.5 M Tris–HCl, pH 8.8 Ni-NTA binding buffer: PBS containing 0.5 M NaCl and 5 mM imidazole, pH 7.4 Ni-NTA wash buffer: PBS containing 0.5 M NaCl and 20 mM imidazole, pH 7.4 Ni-NTA elution buffer: PBS containing 0.5 M NaCl and 200 mM imidazole, pH 7.4 Ni-NTA stripping buffer: PBS containing 0.5 M NaCl and 50 mM EDTA Tris(2-carboxyethyl)phosphine HCl (TCEP) 2.3 Equipment and software AKTA FPLC with UPC-900 and P-920 (GE Healthcare, Little Chalfont, UK).

Techniques: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy