Profiling protein-binding glycosaminoglycan oligosaccharides using a simple label-free electrophoretic assay

Abstract

Glycosaminoglycans (GAGs) interact with numerous proteins to regulate key biological processes such as coagulation, cell migration, and growth factor signaling. Despite their biological importance, mapping these interactions and identifying the structural determinants that govern GAG-protein recognition remains analytically challenging, particularly in complex or polydisperse systems. Here, we introduce a simple, label-free carbohydrate polyacrylamide gel electrophoresis (C-PAGE) approach that visualizes GAG-protein complex formation through binding-induced signal suppression of GAG bands. Instead of tracking the mobility of protein-GAG complexes, as it is classically performed in gel-shift assays, C-PAGE directly monitors the disappearance of the free GAG bands stained with Stains-All. Using model high-affinity heparin-binding proteins, the chemokine stromal cell-derived factor 1 α (SDF-1α) and the basic fibroblast growth factor (FGF-2), we demonstrate that C-PAGE clearly distinguishes specific interactions with heparin oligosaccharides from non-binding controls. The method was successfully extended to a micromolar-affinity heparin-binding protein, the interleukin 8 (IL-8), and to complex mixtures such as low-molecular-weight heparins (LMWH), revealing the preferential disappearance of highly sulfated and/or longer GAG species. Competitive assays further enabled qualitative ranking of GAG binding affinities. Finally, the selective interaction of antithrombin III with 3-O-sulfated motifs was unambiguously detected, underscoring the remarkable sensitivity of C-PAGE to fine structural modifications. Altogether, C-PAGE provides a rapid, visual, and cost-effective screening tool to assess GAG-protein binding specificity and structure-activity relationships, complementing advanced biophysical and structural methods in fundamental and applied glycobiology.

Keywords: carbohydrate polyacrylamide gel electrophoresis; heparin; label-free detection; protein–glycosaminoglycan interactions; structure–activity relationships.