Efficient N-Glycan Profiling from Cell Lysates

Whole cell lysates were prepared from MEFs collected by lysing in Tris-buffered saline (TBS) (50 mM tris(hydroxymethyl)aminomethane, 150 mM NaCl, pH 7.4), containing 1% Triton X-100, 1 mM EDTA, and a protease inhibitor cocktail (cOmplete^TM, Roche, Basel, Switzerland) as described previously [104 (link),105 (link)]. N-glycans were released from the whole-cell lysate protein (25 μg) with 2 units of PNGase F (Roche) after reductive alkylation and trypsin digestion. The released N-glycans were captured and labeled with N^α-((aminooxy)acetyl)tryptophanylarginine methyl ester (aoWR) by BlotGlyco beads (Sumitomo Bakelite, Tokyo, Japan) as described previously [106 (link),107 (link)]. After removal of excess reagents by MassPrep HILIC μElution plate, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analyses of aoWR-labeled glycans were performed on Autoflex Speed (Bruker Daltonics, Billerica, MA, USA) operated in positive-ion reflector mode. For MS acquisition, aoWR-labeled glycans in acetonitrile were mixed 1:1 with dihydrobenzoic acid (10 mg/mL in 50% acetonitrile) and spotted on the target plate.