O-glycosylation is a highly diverse and complex form of protein 
post-translational modification. Mucin-type O-glycosylation is initiated by the 
transfer of N-acetyl-galactosamine (GalNAc) to the hydroxyl group of serine, 
threonine and tyrosine residues through catalysis by a family of 
glycosyltransferases, the UDP-GalNAc:polypeptide 
N-acetylgalactosaminyltransferases (E.C. 2.4.1.41) that are conserved across 
metazoans. In the last decade, structural characterization of glycosylation has 
substantially advanced due to the development of analytical methods and advances 
in mass spectrometry. However, O-glycosite mapping remains challenging since 
mucin-type O-glycans are densely packed, often protecting proteins from cleavage 
by proteases. Adding to the complexity is the fact that a given glycosite can be 
modified by different glycans, resulting in an array of glycoforms rising from 
one glycosite. In this study, we investigated conditions of solid phase 
extraction (SPE) enrichment, protease digestion, and Electron-transfer/Higher 
Energy Collision Dissociation (EThcD) fragmentation to optimize identification 
of O-glycosites in densely glycosylated proteins. Our results revealed that 
anion-exchange stationary phase is sufficient for glycopeptide enrichment; 
however, the use of a hydrophobic-containing sorbent was detrimental to the 
binding of polar-hydrophilic glycopeptides. Different proteases can be employed 
for enhancing coverage of O-glycosites, while derivatization of negatively 
charged amino acids or sialic acids would enhance the identification of a short 
O-glycopeptides. Using a longer than normal electron transfer dissociation (ETD) 
reaction time, we obtained enhanced coverage of peptide bonds that facilitated 
the localization of O-glycosites. O-glycosite mapping strategy via proteases, 
cut-off filtration and solid-phase chemoenzymatic processing. Glycopeptides are 
enriched by SPE column, followed by release of N-glycans, collection of higher 
MW O-glycopeptides via MW cut-off filter, O-glycopeptide release via O-protease, 
and finally detected by LC-MS/MS using EThcD.