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Dae Sik Cho,Myung Jin Oh,Hyun Joo An 한국당과학회 2022 한국당과학회 학술대회 Vol.2022 No.07
Keratan sulfate (KS)-proteoglycan is a linear polymer consisting of repeating disaccharide with a negative charge imparted by sulfate. KS is a sophisticated molecule with a diverse structure, and unique functional roles in the cornea, cartilage catabolism, and joint formation. In particular, accumulation of undegraded KS by enzyme deficiency results in skeletal cartilage dysplasia and lysosomal storage disorders such as mucopolysaccharidosis IVA and IVB. Therefore, detection and quantitation of KS are important for the diagnosis and treatment of related diseases, but it is still analytically challenging due to low concentration of KS in biological samples and the difficulty of selective separation of KS. In this study, we developed a highly sensitive and selective method for quantitating KS in mouse urine and liver using the combination of solid-phase extraction (SPE) and LC-MS/MS MRM. In addition, the Folch method and spin column were used to efficiently remove liver lipids and urine metabolites, respectively. Thereafter, KS was selectively fractionated by SPE with a PGC cartridge according to its molecule size and polarity (acidity, pKa). Quantitation using LC-MS/MS MRM were optimized and validated by polymer KS, LacNAc(1S), LacNAc(2S) standards and Keratanase II prior to actual sample analysis. In the case of urine, KS was quantified in the range of 1-20 pg/mL, and in the liver, it was quantified in the range of 0.1-20 pg/mg. Our new platform with high sensitivity and selectivity can be widely used for diagnosis and treatment of KS-related disease.