Site-specific characterisation of mucin-type O-linked glycosylation is an 
analytical challenge due to glycan heterogeneity, lack of glycosylation site 
consensus sequence and high density of occupied glycosylation sites. Here, we 
report the use of electron transfer dissociation (ETD) for the site-specific 
characterisation of densely glycosylated mucin-type O-linked glycopeptides using 
ESI-IT-MS/MS. Synthetic glycopeptides from the human mucin-1 (MUC-1) tandem 
repeat region containing a range of O-linked, tumour-associated carbohydrate 
antigens, namely Tn, T and sialyl T, with different glycosylation site 
occupancies and an increasing number of tandem repeats were studied. In 
addition, a glycopeptide from the anti-freeze glycoprotein of Antarctic and 
Arctic notothenoids, bearing four O-linked, per-acetylated T antigens was 
characterised. ETD MS/MS of infused or capillary LC-separated glycopeptides 
provided broad peptide sequence coverage (c/z·-type fragment ions) with intact 
glycans still attached to the Ser/Thr residues. Thus, the glycosylation sites 
were unambiguously determined, while simultaneously obtaining information about 
the attached glycan mass and peptide identity. Highly sialylated O-glycopeptides 
showed less efficient peptide fragmentation, but some sequence and glycosylation 
site information was still obtained. This study demonstrates the capabilities of 
ETD MS/MS for site-specific characterisation of mucin-type glycopeptides 
containing high-density O-linked glycan clusters, using accessible and relative 
low-resolution/low-mass accuracy IT MS instrumentation.