O-glycosylation is another important protein glycosylation modification besides N-glycosylation. Unlike N-glycosylation, O-glycan chains are usually attached to serine (Ser) or threonine (Thr) residues. O-glycosylation site analysis typically involves multiple steps, including protein extraction, proteolytic digestion, enrichment of O-glycosylated peptides, and liquid chromatography-mass spectrometry analysis.