Heparan Sulfate/Heparin Analysis Service

What Does Heparin and Heparan Sulfate Do?

Heparin (HP)/heparan sulfate (HS), a polyanion and heteropolysaccharide, belongs to the glycosaminoglycan family. HP/HS is ubiquitously present on the cell surface and extracellular matrix (ECM), as well as in the intracellular environment (mast cells). Since its discovery in 1916, HP and its low-molecular preparations, one of the most important anticoagulants, have been widely used in clinical treatment. The various structural features of HP/HS allow these molecules to interact with various protein (e. g., enzyme inhibitor, chemokine, growth factor, morphogen and other signaling proteins) to participate in various physiological and pathological processes such as coagulation, cell adhesion, inflammation, cell migration, differentiation, and even pathogen infection. These important biological effects have aroused great attention in the structural and functional studies and clinical applications of HP/HS.

The backbone of HP/HS polysaccharides is composed of repeating disaccharide units consisting of D-glucuronic acid (GlcA)/L-iduronate acid (IdoA) and N-acetyl-D-glucosamine (GlcNAc). During the biosynthesis of HP/HS, their repeated disaccharides, which initial the synthesis of a common precursor consisting of the units of GlcA-GlcNAc, followed by further modification by several enzymes, including N-deacetylase-N-sulfo and 3- and 6-sulfo, which modify GlcNAc residues; A C5- epimerase that converts GlcA to an IdoA residue; And 2-O- sulfotransferase to catalyze the sulfation of GlcA/IdoA residues. These modifications result in variation of disaccharide in HP/HS polysaccharides, making HP/HS the most complex polymer in nature. Therefore, the high structural complexity seriously hinders the structural and functional research of HP/HS.

Fig 1. Biosynthesis of heparin/heparan sulfate and chondroitin sulfate. A. synthesis of tetrasaccharide linker region; B. the polymerization and modification pathway of heparin/heparan sulfate; C. the polymerization and modification pathway of chondroitin sulfate.

Metabolite Analysis Content in Creative Proteomics

Heparan sulfate and heparin consist of repeating disaccharide units, composed of glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc), with varying degrees of sulfation and epimerization. Understanding the structure and modifications of HS/heparin is crucial for elucidating their functions and mechanisms of action.

• Comprehensive HS/heparin analysis involves the identification and quantification of specific metabolites, such as:
• Disaccharide analysis: Determining the disaccharide composition of HS/heparin provides insights into the types and proportions of disaccharide units present, enabling structural characterization and comparison between samples.
• Sulfation pattern analysis: Sulfation is a critical modification in HS/heparin that influences its biological activity. Analyzing the sulfation pattern helps to understand the specific roles played by different sulfation patterns in various physiological and pathological processes.
• Epimerization analysis: Epimerization refers to the interconversion of GlcA to iduronic acid (IdoA) residues. Determining the distribution of GlcA and IdoA residues within HS/heparin molecules helps to elucidate the structural heterogeneity and functional implications of these modifications.
• Size analysis: HS and heparin molecules exhibit considerable size heterogeneity due to variations in chain length. Analyzing the size distribution provides valuable information about the molecular weight distribution and polydispersity of HS/heparin samples.

Platform for Heparin and Heparan Sulfate Assay

Liquid Chromatography-Mass Spectrometry (LC-MS):

Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer is a high-performance hybrid quadrupole-orbitrap mass spectrometer. It offers exceptional sensitivity, resolution, and mass accuracy, making it suitable for the detailed analysis of HS/heparin metabolites.

Gel Permeation Chromatography-Mass Spectrometry (GPC-MS):

ACQUITY™ Ultra Performance Convergence Chromatography™ (UPC2) System provides a unique chromatographic separation technique that enables efficient separation of HS/heparin samples based on size. The combined GPC-MS approach allows for the characterization of the size distribution and polydispersity of HS/heparin molecules.

High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS):

The Agilent 1290 Infinity II LC system, combined with the Agilent 6550 iFunnel Q-TOF LC/MS, provides high-resolution and accurate mass measurements for HS/heparin analysis. It offers excellent sensitivity and selectivity, enabling the identification and quantification of HS/heparin metabolites.

Capillary Electrophoresis-Mass Spectrometry (CE-MS):

The SCIEX CESI 8000 Plus CE system coupled with a mass spectrometer offers high-resolution separation and sensitive detection for HS/heparin analysis. The combination of CE and MS allows for the analysis of disaccharide units, sulfation patterns, and epimerization states in HS/heparin samples.

Fig 2. Figure A shows a work-flow for performing disaccharide composition analysis with three steps. The recover calibrators are added in that heparan sulfate extraction stage or step 1; A disaccharide calibrator is added in the heparin lyase digestion stage or step 2; A disaccharide calibrator was mixed with various concentrations of unlabeled disaccharide and analyzed by LC-MS/MS in analysis stage or step3. Figure B shows the ion chromatograms of the three isotope-labeled formats disaccharide from MRM. Figure C shows the dynamic range of analysis in the presence of a disaccharide calibrator.

Sample requirement

• Serum/plasma: 500 μl/sample

Repeated freezing and thawing of samples must be avoided. The serum sample should be precipitated in the collection tube for 30 minutes at room temperature, then transported to the centrifuge tube and centrifuged at 8000 rpm for 5 minutes. After centrifugation, the supernatant was equally divided into a freezing tube of 500 uL / sample.

• Protein: 100 µg
• Anticoagulated blood (EDTA): 1 mL

Anticoagulants and preservatives must be added immediately after collection and then frozen at -80 °C.

• Urine: 1 ml/sample

Urine samples should be equally divided into centrifuge tubes with 1 mL per tube, each tube is added with 1/100 (w/v) sodium azide and stored at -80 °C.

• Animal tissues: 200 mg/sample

Samples should be frozen in liquid nitrogen immediately and then transported to -80 °C for storage after collected.

• Cells: ≥ 1 × 10⁷/sample

Cytoactive should be terminated immediately to maintaining cell integrity.

• Feces: 500 mg/sample

In general, to assure enough sample to fulfill the whole project, the volume of the single sample need to be offered as much as possible. The remaining samples will be stored for one year free of charge and returned at any time if necessary. All samples need to be stored and transported at -80°C and try to avoid using surfactants (SDS, Triton-X) and inorganic salts.

Clinical samples are repeated in no less than 30 cases in a single group.

Animal samples are repeated in no less than 9 cases in a single group.

Report delivery

• Experimental procedure
• Parameters of HPLC and MS
• Raw data, chromatograms and mass spectra
• Metabolites quantification data
• Custom analysis report

Service cycle

• Sample testing: 5-10 working days
• Data analysis: 5-10 working days

Creative Proteomics metabolism analysis platform is committed to the all-around, reliable and accurate analysis service for a variety of target substances, which is suitable for life-science research, drug exploration, biological determination and other fields. We sincerely hope to cooperate with you to assistant your scientific research.

References

1. M. Suflita, L. Fu, W. He, et al. Heparin and related polysaccharides: synthesis using recombinant enzymes and metabolic engineering. Appl Microbiol Biotechnol, 2015, 99(18):7465-79.
2. K. Sugahara, H. Kitagawa. Heparin and heparan sulfate biosynthesis. IUBMB Life, 2002, 54(4):163-75.
3. W. Wang, L. Shi, Y. Qin, et al. Research and Application of Chondroitin Sulfate/Dermatan Sulfate-Degrading Enzymes. Front Cell Dev Biol, 2020, 8:560442.
4. Z. Wang, K. Arnold, Y. Xu, et al. Quantitative analysis of heparan sulfate using isotopically labeled calibrants. Commun Biol, 2020, 3(1):425.