N-glycans in Toxicodendron vernicifluum lacquer laccase

Abstract The N-glycans in Toxicodendron vernicifluum ( Rhus vernicifera ) lacquer laccase was elucidated for the first time through a combination of enzymatic digestion and subsequent mass spectrometry measurements using LC–MS/MS and MALDI–TOF MS. Lacquer laccase was isolated from a Japanese lacquer acetone powder from consecutive Sephadex C-50 and DEAE A-50 column chromatography. Trypsin and chymotrypsin digestions of the lacquer laccase resulted in a mixture of peptides and N-glycopeptides, which were treated with peptide- N -glycosidases and then N α -(aminooxyacetyl)tryptophanylarginine methyl ester (aoWR) to give the aoWR-labelled N-glycans. The MS measurements revealed that GlcNAc 4 Hex 5 Fuc 3 Xyl 1 N-glycan was attached at 12 N-glycosylation sites (Asn 5, 14, 180, 194, 233, 274, 284, 347, 364, 381, 398, and 519), GlcNAc 3 Hex 4 Fuc 2 Xyl 1 N-glycan at two sites (Asn 124 and 454), and GlcNAc 3 Hex 6 Fuc 1 Xyl 1 N-glycan at one site (Asn 28). A database search (Mascot search) of the peptides also suggested the presence of N-glycans at the 15 potential N-glycosylation sites (Asn-X-Ser/Thr).