Comparative Quantification Method for Glycosylated Products Elongated on β-Xylosides Using a Stable Isotope-Labeled Saccharide Primer.
Yuya OtsukaToshinori SatoPublished in: Analytical chemistry (2018)
The structures and amounts of glycosaminoglycan (GAG) produced by cells have attracted much interest because GAG biosynthesis activity can change in cellular processes such as disease and differentiation. β-Xylosides, also called saccharide primers, have been used as artificial acceptors not only to generate GAG oligosaccharides in cells and tissues but also to investigate their biosynthetic pathways. Various analytical methods have been applied to confirm the structure and amounts of GAG oligosaccharides elongated using saccharide primers, yet sample preparation processes such as solid-phase extraction in analysis can cause experimental error and disrupt accurate comparative quantification of glycosylated products. In this study, we developed a new quantification method using a deuterium-labeled saccharide primer. The "heavy" and "light" primers were chemically synthesized, and priming abilities were confirmed by liquid chromatography-tandem mass spectrometry. Relative peak areas of light/heavy products showed good linearity and were well correlated with the theoretical amounts of glycosylated products. Then, as a validation study, we carried out a biosynthesis inhibition assay using known GAG biosynthesis inhibitors. According to the relative quantification using saccharide primers, differences in the mode-of-action among the four GAG biosynthesis inhibitors were dependent on the GAG biosynthetic pathway. Our results indicate that the method will likely forge a new path for comparative glycosaminoglycomics using cultured cells and tissues.
Keyphrases
- liquid chromatography tandem mass spectrometry
- induced apoptosis
- solid phase extraction
- cell cycle arrest
- simultaneous determination
- gene expression
- molecularly imprinted
- ms ms
- high performance liquid chromatography
- cell wall
- high resolution
- oxidative stress
- mass spectrometry
- signaling pathway
- cell death
- liquid chromatography
- endoplasmic reticulum stress
- tandem mass spectrometry
- high throughput
- gas chromatography mass spectrometry
- pet imaging
- solar cells