A Mass Spectrometry Method for the Quantification
of Heparan Sulfate in Juniper in Modern Applications of Bioequivalence & Bioavailability (MABB)
Heparan sulfate proteoglycans (HSPG) are present on the cell surface as well extracellular matrix, where they interact with a series of ligands [1]. Like most other macromolecules, HSPG are processed within the lysosome. However, in cases of lysosomal enzyme deficiency, HSPG are not properly digested leading to accumulation of these HSPG and disease state known as mucopolysaccharidoses [2]. We have developed a robust method for the analysis of heparan sulfate from cell extracts by measuring heparan lyase-digested disaccharides. For normalization, the samples were spiked with an internal standard to correct for losses during sample preparation which involved sample clean-up using hypercarb SPE columns. The disaccharide abundances were determined by LC-MS/MS using a triple quadruple mass spectrometer set-up in selected reactions monitoring mode. We applied this method to measure the relative heparan sulfate disaccharide abundances in three mutants and three wild type cell extracts. The disaccharide amounts in the mutant cells were determined to be approx. 6-7 fold higher than those in wild type cells.
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