Glycopeptides, isolated from a trypsinolysate of functional unit (FU) RtH2-e of Rapana thomasiana hemocyanin subunit 2, were analysed by electrospray ionization mass spectrometry and MS/MS. From the molecular mass observed after deglycosylation, it was inferred that all glycopeptides shared the same peptide stretch 92 143 of FU RtH2-3 with a glycosylation site at Asn-127. Besides the core structure Man(3)GlcNAc(2) for N-glycosylation, structures with a supplementary GlcNAc linked to either the Man(α 1 3) or the Man(α 1 6) arm and/or an additional tetrasaccharide unit connected to the other Man arm were observed. indicating the existence of microheterogeneity at the glycan level. The tetrasaccharide unit contains a central fucose moiety substituted with 3-O-rnethylgalactose and N-acetylgalactosamine. and linked to GlcNAc at the reducing end. This structure represents a novel N-glycan motif and is likely to be immunogenic. A second potential site for N-glycosylation in FU RtH2-e at Asn-17 was shown to be not glycosylated. © 2005 Elsevier Inc. All rights reserved.