Updated publication reference for PubMed record(s): 30824935. Here, using LC-MS/MS, we describe for the first time a linkage region trisaccharide comprising [-GlcUA-Gal-Xyl-O-] as an alternative point of entry for glycosaminoglycan (GAG) biosynthesis. The trisaccharide was identified in the proteoglycan bikunin from urine of human healthy donors present as a disulfated pentasaccharide, [deltaHexUA-GalNAc(S)-GlcUA-Gal(S)-Xyl-O-] after chondroitinase ABC degradation. Furthermore, it was identified in chondroitin sulfate primed on xylosides isolated from human cell lines, present as the corresponding disulfated pentasaccharide after chondroitinase ABC degradation.