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Development of a specific and sensitive LC-MS/MS method to quantify Heparan Sulfate 3-O-Sulfotransferase-1 activity.

Vivien Uyen Doan1, Yongmei Xu1, Zhangjie Wang2

  • 1Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, United States.

Glycobiology
|March 31, 2026
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Summary

A new LC-MS/MS assay quantifies heparan sulfate 3-O-sulfotransferase-1 (3OST-1) activity, crucial for Alzheimer's and cancer. This sensitive method enables biomarker development and therapeutic targeting for HS-related diseases.

Keywords:
3-OsulfationLC–MSenzyme activity assayheparan sulfatesulfotransferase

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Area of Science:

  • Biochemistry
  • Glycobiology
  • Molecular Biology

Background:

  • Heparan sulfate (HS) is a critical glycan regulating biological processes via specific sulfation patterns.
  • 3-O-sulfation, catalyzed by heparan sulfate 3-O-sulfotransferase-1 (3OST-1), is vital for anticoagulation, Alzheimer's disease, and cancer progression.
  • Existing assays lack the sensitivity and specificity for direct measurement of 3OST-1 activity in biological systems.

Purpose of the Study:

  • To develop a sensitive and specific assay for quantifying 3OST-1 activity.
  • To utilize the assay for studying the functional impact of the 3OST-1 E86Q mutant.
  • To establish a quantitative tool for 3OST-1 activity and explore therapeutic strategies.

Main Methods:

  • Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay.
  • Utilized a structurally defined HS hexasaccharide substrate and 13C-labeled internal standards.
  • Employed heparinase digestion and AMAC derivatization for product detection.

Main Results:

  • The LC-MS/MS assay achieved nanogram-level detection with 100-fold improved sensitivity over HPLC.
  • Successfully quantified endogenous 3OST-1 activity in HCT-116 cells and conditioned media.
  • Confirmed the 3OST-1 E86Q mutant as a dominant-negative inhibitor, reducing 3-O-sulfated HS products by over 80% without affecting overall HS levels.

Conclusions:

  • The developed LC-MS/MS assay is the first quantitative tool for measuring 3OST-1 activity.
  • The study establishes a dominant-negative strategy for 3OST-1 functional studies.
  • This assay opens avenues for biomarker development and therapeutic targeting in HS-related pathologies.