Filter-entrapment enrichment pull-down assay for glycosaminoglycan structural characterization and protein interaction

Yanlei Yu, Fuming Zhang, Gina Renois-Predelus, I. Jonathan Amster, Robert J. Linhardt

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

Heparins are the most pharmaceutically important polysaccharides. These heparin-based anticoagulant/antithrombotic agents include unfractionated heparins, low molecular weight heparins (LMWHs) and ultralow molecular weight heparins (ULMWHs). Heparins exhibit their pharmacological and biological activities through interaction with heparin-binding proteins. The prototypical heparin-binding protein is antithrombin III (AT), responsible for heparin's anticoagulant/antithrombotic activity. This study describes a filter-trapping method to isolate the chains in enoxaparin, a LMWH, which bind to AT. We demonstrate this method using the ULMWH, fondaparinux, which consists of a single well defined AT binding site. The interacting chains of enoxaparin are then characterized by activity assays, top-down liquid chromatography-mass spectrometry, and capillary zone electrophoresis mass spectrometry. This filter-trapping assay is an improvement over affinity chromatography for isolating heparin chains interacting with heparin binding proteins.

Original languageEnglish
Article number116623
JournalCarbohydrate Polymers
Volume245
DOIs
StatePublished - 1 Oct 2020
Externally publishedYes

Keywords

  • Antithrombin III
  • Capillary electrophoresis
  • Filter trapping
  • Heparin
  • Heparin-binding proteins
  • Mass spectrometry

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