Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 5201-5208 |
| Number of pages | 8 |
| Journal | Analytical Chemistry |
| Volume | 90 |
| Issue number | 8 |
| DOIs | |
| Publication status | Published - 2018 Apr 17 |
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ASJC Scopus subject areas
- Analytical Chemistry
Cite this
Comparative Quantification Method for Glycosylated Products Elongated on β-Xylosides Using a Stable Isotope-Labeled Saccharide Primer. / Otsuka, Yuya; Sato, Toshinori.
In: Analytical Chemistry, Vol. 90, No. 8, 17.04.2018, p. 5201-5208.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Comparative Quantification Method for Glycosylated Products Elongated on β-Xylosides Using a Stable Isotope-Labeled Saccharide Primer
AU - Otsuka, Yuya
AU - Sato, Toshinori
PY - 2018/4/17
Y1 - 2018/4/17
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=85045681252&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.7b05438
DO - 10.1021/acs.analchem.7b05438
M3 - Article
C2 - 29533603
AN - SCOPUS:85045681252
VL - 90
SP - 5201
EP - 5208
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 8
ER -