The identified O-glycopeptides , glycan com position, fragmentation technique, annotation technique (software/manual), and glycosylation sites are listed in Table I (and in supplemental Table S1)
Regions of glycosylation sites identified via CID and ETD both before and after partial de-glycosylation are shown in Fig. 4A
The identified O-glycopeptides characterized 168 glycosylation sites
This indicated that 63% of the identified Ser/Thr residues (266 Ser/Thr , both glycosylated and non-glycosylated, were identified) in lubricin were O-glycan modified (Fig. 4B) with a bias toward Thr glycosylation due to the high Thr content (supplemental Fig
S2)
An extended STP-rich region was also apparent spanning amino acids 232–1056, larger than the previously defined mucin-like domain suggested in UniProt
The entire lubricin molecule has in total 370 potential O-glycosylation sites
Of these, 35% of Ser/Thr (130 glycans) were HexNAc (GalNAc) modified with a distribution throughout the extensively glycosylated STP-rich region (Fig. 4B)
Core 1 modified (43%, 161 glycans) and larger core 2 modified (23%, 85 glycans) glycopeptides were also identified (Fig. 4B)
The high-abundant core 1 modified Ser/Thr were uniformly distributed throughout the STP-rich region , whereas the low-abundant core 2 modified residues were limited to the previously defined mucin-like domain (aa 348–856) (Fig. 4B)
This was likely because the accumulative nature of glycopeptides from the repeat region made them easier to detect, and it might not necessarily be a reflection of core 2 enrichment in the repeat area
Outside the STP region , only a single GalNAc-modifiedThr (1159NGTLVAFR1166) was identified
This residue , in the hemopexin 1 domain (1148–1191), was also shown to be glycosylated with core 2 structures (Figs. 4A and 4B; supplemental Fig
S1; supplemental Table S2)
The identification of core 2 glycopeptides confirmed the presence of core 2 structures on lubricin
The identification of core 2 together with single HexNAc (GalNAc)-modifiedglycopeptides suggested that lubricin glycosylation might also have other roles in addition to lubrication
The majority of the GalNAc extended into either core 1 or core 2 sialylated structures (73 glycans; Fig. 4B)
Both mono- and disialylated core 1 and core 2 modified Ser/Thr were identified (Table I and supplemental Table S1), but monosialylation was more prevalent, which is consistent with previously identified synovial lubricin O-glycans (38)