The human KNG1 gene codes for two splicing variants of kininogen, namely low-molecular and high-molecular weight kininogen
The latter is involved in blood coagulation and the assembly of the kallikrein-kinin system and was identified in the present study by six O-glycopeptides
Currently nine O-glycosylation sites/regions are described in literature for kininiogen-1—presumably all being decorated with mucin-type core 1 or possibly core 8 O-glycans (30, 60–62)
Experimental glycoproteomic evidence on the macro and microheterogeneity of kininogen-1 is still missing, though
In the present study, four kininogen-1 O-glycosylation sites , including one novel site ( Ser604 ), could be pinpointed and described with respected to the com position of the attached O-glycans (Table I)
The identified O-glycopeptide 600FNPIDFPD610 (m/z 734.263+) carries a disialylated T-antigen and harbors three potential O-glycosylation sites
ETD analysis implies the occupancy of Ser604 , because of the presence of a signal at m/z 1638.301+, corresponding to a c6 ion (supplemental Fig
S6)
Also of note, in previous studies the use of trypsin did not allow to pinpoint occupied O-glycosylation sites in the region aa119–161 (30, 61)
Proteinase K , however, generated two distinct O-glycopeptides (132EGPVVA138m/z 664.702+, 146VHPIQ152m/z 719.232+) that allowed pinpointing the site Thr137 and the region Ser150/Thr151
For the latter, unfortunately, the ETD spectrum quality did not allow localizing the exact site
The peptide 132EGPVVA138 (m/z 664.702+) could not be identified correctly by MASCOT database search, because of missing fragment ions
However, the peptide could be identified via manual de novo annotation supported by mass tag ([283.0 Da]VVTA) assisted de novo sequencing using the tool MS-Homology (http ://prospector.ucsf.edu/prospector) (supplemental Fig
S5)
The peptide identity was further verified by the identification of the glycosylated peptide 132EGPVVAQ138 (m/z 874.282+) in a subsequent HILIC fraction (supplemental Fig