Simultaneous Analysis of Heparan Sulfate, Chondroitin/Dermatan Sulfates, and Hyaluronan Disaccharides by Glycoblotting-Assisted Sample Preparation Followed by Single-Step Zwitter-Ionic-Hydrophilic Interaction Chromatography

Abstract

Glycosaminoglycans (GAGs) play important roles in cell adhesion and growth, maintenance of extracellular matrix (ECM) integrity, and signal transduction. To fully understand the biological functions of GAGs, there is a growing need for sensitive, rapid, and quantitative analysis of GAGs. The present work describes a novel analytical technique that enables high throughput cellular/tissue glycosaminoglycomics for all three families of uronic acid-containing GAGs, hyaluronan (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), and heparan sulfate (HS). A one-pot purification and labeling procedure for GAG Δ-disaccharides was established by chemo-selective ligation of disaccharides onto high density hydrazide beads (glycoblotting) and subsequent labeling by fluorescence. The 17 most common disaccharides (eight comprising HS, eight CS/DS, and one comprising HA) could be separated with a single chromatography for the first time by employing a zwitter-ionic type of hydrophilic-interaction chromatography column. These novel analytical techniques were able to precisely characterize the glycosaminoglycome in various cell types including embryonal carcinoma cells and ocular epithelial tissues (cornea, conjunctiva, and limbus).

Total cellular glycomics allows characterizing cells and streamlining the discovery process for cellular biomarkers

“…The streamlined GAG and GSL extraction procedures were essentially the same as previously described (12,16). Detailed procedures and materials are provided in SI Appendix, Materials and Methods.…”

A review of bioactive plant polysaccharides: Biological activities, functionalization, and biomedical applications

“…Understanding of this structure-activity relationship of heparin offers the opportunity to develop drugs with higher specificity and better regulation of coagulation and to explore other therapeutic applications such as infection, inflammation, cancer and wound-healing treatment (Linhardt, 2003;Rajangam et al, 2006;Sakiyama-Elbert, 2014;Zhang et al, 2013a;Zhang, Wardwell, & Bader, 2013b). CS/DS chains are composed variably sulfated N-acetylgalactosamine and glucuronic acid or its epimeride iduronic acid disaccharides repeating units (Silbert & Sugumaran, 2002;Takegawa et al, 2011). Growing research evidence reveals the previously less or B i o a c t i v e C a r b o h y d r a t e s a n d D i e t a r y F i b r e 5 ( 2 0 1 5 ) 3 1 -6 1 unperceived functions of CS/DS (Sugahara et al, 2003), for example being a key molecule of the brain extracellular matrix (Kwok, Warren, & Fawcett, 2012), regulating the cell adhesion, proliferation and migration during wound healing (Zou et al, 2004), signaling of growth factor in skeleton (Alliston, 2010).…”

Quantitative analysis of glycosaminoglycans, chondroitin/dermatan sulfate, hyaluronic acid, heparan sulfate, and keratan sulfate by liquid chromatography–electrospray ionization–tandem mass spectrometry

“…To date, the quantitative compositional analysis of GAG disaccharides has been performed by the ultraviolet (UV) method [13,14], fluorescence [14,15], and mass spectrometry (MS) [16]. The closely related, but subtly different structures of various GAGs have complicated their detailed and extensive analysis.…”