Isolation and Compositional Analysis of Glycosaminoglycans

Abstract

The compositional and structural analysis of GAGs is challenging due to their heterogenous structures. Strong anion exchange (SAX) HPLC can aid in the compositional analysis of GAGs and can separate complex mixtures based on charge and degree of sulfation. Herein we describe the digestion and release of GAGs from tissue, and the compositional analysis using SAX-HPLC.

1. Introduction

Glycosaminoglycans (GAGs) are linear polysaccharides composed of repeating disaccharide units of a uronic acid and a hexosamine [1, 2]. GAGs can be N-acetylated and highly O- or N-sulfated, with up to three sulfates per disaccharide unit [3]. GAG biosynthesis is a non-template driven enzymatic process; therefore, GAGs are extremely heterogeneous in structure. Analytical techniques such as X-ray diffraction, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) are used to determine the composition and structure of GAGs [4]. Due to the generally low quantity of GAGs derived from biological processes, mass spectrometry has emerged as a powerful tool for GAG analysis [5–13].

Separation techniques have been heavily utilized in the various characterization methods of GAGs, and GAG fragments. These techniques paired to either an MS instrument or a UV detector can yield compositional information. HPLC techniques are used to separate GAG mixtures according to their degrees of polymerization (dp) [4, 14–16]. Size-exclusion chromatography (SEC) is a robust and simple technique that can separate chain lengths, mostly in increments of dp 2. SEC is a great technique for determining profiles of compositions in a mixture, but this technique cannot separate isomers (i.e. GAGs with the same number of sulfate groups, but at different positions) [17, 18]. An ion pairing reagent can be used for reverse-phase ion pairing (RPIP), which can separate similar GAGs [19, 20]. This technique suffers from the difficulty to remove ion pairing reagent from a mass spectrometer once it has been introduced.

Strong anion exchange (SAX) HPLC separates based on charge and can distinguish different degrees of sulfation. Prior to SAX analysis, a GAG sample is digested into its (mostly) disaccharide building blocks by an enzyme or a group of enzymes. For example, heparan sulfate (HS) is digested by heparinases I, II and III, which cleave HS between the glucosamine and uronic acid, resulting in the formation of a double bond between C4 and C5 of the uronic acid. This double bond allows for UV detection of the saccharides [6, 18, 21] after the SAX separation. Within this chapter we outline the methods to digest, derivatize and analyze HS mixtures using SAX-HPLC (Figure 1).

Figure 1.

Experimental flow-chart for determining GAG composition using SAX-HPLC. A) Extraction of GAGs from whole cells and tissue, B) isolation and purification of GAGs, and C) GAG-specific digestion and analysis.

The utility of these separation techniques is highlighted in the analysis of GAG-based biotherapeutics, such as low-molecular weight heparins (LMWHs). LMWHs, which are derived from heparin by controlled depolymerizations by chemical or enzymatic means, have multiple clinical uses and require stringent methods of characterization to determine batch-to-batch and vendor-to-vendor consistency.

2. Materials

1. All solutions should be prepared in ultrapure grade water (18 MΩ-cm at 25°C) and analytical grade solvents and reagents.

2. Homogenization, and Protein/Nucleic Acid Digestion

   1. Mortar and pestle, or appropriate homogenization equipment.
   2. Acetone (analytical grade, CAS 67-64-1).
   3. Isopropyl ether (analytical grade, CAS 108-20-3).
   4. Pronase digestion buffer: 0.1 M Tris-HCl, pH 8.0 (CAS 1185-53-1), 2 mM CaCl₂ (CAS 10043-52-4), 1% Triton X-100 (9036-19-5). Prepare 100 mL of buffer by combining 10 mL of 1M Tris-HCl stock, 2 mL of a 100 mM CaCl₂ stock, and 1 mL of 1% Triton X-100. Bring to 100 mL and mix well. Do not filter. The buffer can be stored for 1 month at 4 °C.
   5. 100 mM Stock solution MgCl₂ (CAS 7786-30-3). Prepare by weighing 9.5 g of MgCl₂ and bring to 100 mL. The buffer can be stored for 1 month at 4 °C.
   6. Pronase protease from Streptomyces griseus (SKU 10165921001).
   7. Benzonase^® nuclease (CAS 9025-65-4).
3. Anion Exchange Chromatography

   1. Equilibration buffer: 20 mM Tris-HCl, pH 7.5, 0.1 M NaCl (CAS 7647-14-5). Prepare 100 mL of buffer by weighing 0.58 g of NaCl, combining with 2 mL of a 1 M Tris-HCl stock, mixing well, and bringing to a final volume of 100 mL. The buffer can be stored for 1 month at 4 °C.
   2. Elution buffer: 20 mM Tris-HCl pH 7.5, 2 M NaCl. Prepare 100 mL of buffer by weighing 11.7 g of NaCl, combining with 2 mL of a 1 M Tris-HCl stock, mixing well, and bringing to a final volume of 100 mL. The buffer can be stored for 1 month at 4°C.
   3. Disposable chromatography columns (eg BioRad Econo-Pac PT 7321010).
   4. DEAE Sephacel weak anion exchanger resin (Cytiva PT 71050001).
4. β-Elimination

   1. Reduction solution: 2 M NaOH, 10% (w/v) NaBH₄ (CAS 16940-66-2). Prepare 10 mL of solution by weighing 1 g of NaBH₄ and dissolving in a 2 M NaOH solution. Prepare this resolution immediately before use and use fresh for each reduction.
5. Desalting

   1. Disposable PD-10 desalting column with Sephadex G-25 resin (Cytiva PT 17085101).
6. Compositional Analysis of GAGs by GAG Lyase Digestion and SAX-HPLC

   1. 8 GAG disaccharide standards (Galen Lab Cat. HD001-HD008). 2 mM stock solutions of: D2A6 (ΔUA,2S-GlcNAc, 6S), D0A6 (ΔUA-GlcNAc, 6S), D2A0 (ΔUA,2S-GlcNAc), D0A0 (ΔUA-GlcNAc), D2S6 (ΔUA,2S-GlcNS, 6S), D0S6 (ΔUA-GlcNS, 6S), D2S0 (ΔUA,2S-GlcNS), D0S0 (ΔUA-GlcNS)[22]. Structures of these disaccharides are shown in Figure 2.
   2. GAG standard solution. 0.1 mM mixture of all 8 HS standard disaccharides. Mix 10 μL of each stock solution and dilute with water to a final volume of 200 μL.
   3. GAG separation stock buffer. Prepare a 100 mM sodium phosphate solution in water by weighing 12 g of sodium phosphate (monobasic). Bring to 1 L with water and buffer to pH 2.5 with phosphoric acid. The stock solution can be stored up to 1 month at 4 °C.
   4. GAG separation buffer A. 2.5 mM sodium phosphate, pH 3.5. Dilute 25 mL of the GAG Separation Stock buffer to 1 L with water. Degas and filter the solution through a 0.4-µm filter before use.
   5. GAG separation buffer B. 2.5 mM sodium phosphate, 1.2 M sodium chloride pH 3.5. Weigh 70.1 g of sodium chloride and add 25 mL of the GAG separation stock buffer. Dilute to 1 L with water. Degas and filter the solution through a 0.4-µm filter before use.

Figure 2.

Heparan sulfate disaccharide standards used for identifying composition with SAX-HPLC. Nomenclature is shown for each disaccharide using the Lawrence et al. method [22].

3. Methods

1. Homogenization, and Protein/Nucleic Acid Digestion

   1. Weigh a clean, empty 50-mL plastic conical tube for each sample. Rinse with acetone and allow to dry. Homogenize tissue by freezing in liquid nitrogen and grinding with a mortar and pestle (Note 1 and Figure 1A).
   2. Delipidate the sample with acetone (Note 2). Transfer the homogenized material to the conical tube with acetone. Rinse the mortar with 5–10 mL of acetone and transfer to the same conical tube. Rotate the powdered tissue in acetone for 24 hours at 4 °C, then centrifuge at 4000 x g to pellet the powdered tissue and remove the acetone fraction to waste. Add 15 mL of fresh acetone and repeat the process. Add 5 mL of isopropyl ether to the defatted pellet, mix, and rotate for 30 minutes at room temperature (RT). Centrifuge at 4000 x g, remove the ether to waste and allow the defatted powder to dry completely. Reweigh the tube and record the weight of the delipidated dry tissue.
   3. Resuspend delipidated dry powder to a concentration of 100 mg/mL in the pronase digestion buffer. Add pronase at a concentration of 0.8 mg/mL and digest the tissue with mixing for 24 hours at 55 °C in an orbital shaker at 100 rpm. After 24 hours, add a second aliquot of pronase and continue the digestion for another 24 hours. Inactivate the enzyme by heating to 100 °C for 15 minutes.
   4. Adjust the buffer to 2 mM MgCl₂ and add benzonase to a concentration of 25 mU/mL. Incubate for 2 hours at 37 °C, then inactivate the enzyme by heating to 100 °C for 15 minutes.
   5. Pellet any undigested material by centrifugation at 12000 x g for 15 minutes at RT. Transfer the supernatant (containing GAG) to a new tube and measure the volume.
2. Anion Exchange Chromatography

   1. Pour and prepare weak anion exchange DEAE-Sephacel columns. 2 mL of prepared resin is needed for each column. Centrifuge resin for 5 minutes at 3800 x g and decant the storage solution to waste. Add 15 mL of equilibration buffer and rotate for 30 minutes at RT. Centrifuge at 3800 x g and decant the buffer to waste. Add another 15 mL of equilibration buffer and rotate for 1 hour at RT. Pour each column at ~2 mL of bed volume and equilibrate the columns in 10 column volumes (CV) of equilibration buffer. Load the supernatant to the column, allow it to flow through, and reapply it to the column. Wash the column with another 10 CVs of equilibration buffer, then elute the GAG fraction with 3 CVs of elution buffer into 15 mL centrifuge tube (Figure 1B).
3. β-Elimination

   1. To the 6 mL of GAG elution, add 0.7 mL of the reduction solution, confirm that the pH is basic with pH indicator paper, and incubate overnight at 4 °C [23]. Stop the reaction the next day by adding glacial acetic acid dropwise with a pipettor until no bubbles form. Check the pH with a paper indicator to confirm that the pH is neutral. Dry the sample by lyophilization (Note 3 and Figure 1B).
4. Desalting

   1. Prepare a new PD-10 column by discarding the storage liquid and equilibrating the column in 30 mL of water by allowing it to flow through with gravity. Discard the flow through.
   2. Dissolve the dried GAG sample in 2 mL water. A volume difference will be observed because of the high salt content. Measure the total volume and load onto the column. Allow the sample to enter the column and discard the flow through. Add the difference between the measured volume and 2.5 mL to the column after the sample has completely entered the column (Note 4). Discard the flow through. Elute the sample in 3.5 mL of water, collect, and dry the desalted sample by lyophilization.
5. Compositional Analysis of Heparan Sulfate by GAG Lyase Digestion and SAX-HPLC (UV-Detection)

   1. Aliquot a portion of the purified GAG and reconstitute in an appropriate buffer (Note 5) to a final volume of 100 µL. Add a cocktail of heparinase I, II, and III to the solution and incubate overnight at 37 °C. Inactivate the enzyme by heating at 100 °C for 5 minutes. Pellet protein by centrifugation at 12000 x g for 15 minutes and transfer to a sample vial for HPLC analysis. Digestion of heparin/heparan sulfate with bacterial heparinase enzymes results in an unsaturated disaccharide at the C4-C5 at the uronic acid that can be observed with UV detection (232 nm) (Figure 1C).
   2. Prepare a solution of 8 GAG disaccharide standards from heparin (Note 6). Pipette 10 µL of each standard into a centrifuge tube and bring to a final volume of 200 µL, vortex, and transfer to a sample vial for HPLC analysis.

      Time	Buffer A (%)	Buffer B(%)
      0	97%	3%
      60	0%	100%
      65	0%	100%
      75	97%	3%

   3. Equilibrate a strong-anion exchange column for analytical HPLC use. The following parameters are optimized for a Spherisorb SAX column, 5 µm, 4.6 mm X 250 mm (Waters, PSS832715) (Note 7). It is recommended that the appropriate guard column (e. g. Waters, PSS830055) with cartridge also be utilized to protect the column since high concentrations of salts, and biological materials are being used in the analysis procedure. Always use manufacturer’s recommendations for the column of use.
   4. To analyze samples and standards: Inject a 10-µL volume of sample or standard. Run a gradient of 3% to 100% GAG Separation Buffer B over 60 minutes at a flow rate of 1 mL/min. A full instrument program is shown below. Detect at 232 nm.
   5. Identify each disaccharide based on retention time compared to the standard run. Because a double bond is formed from the heparinase enzymes, the 232-nM absorbance signature can be directly related to the concentration of each GAG in solution (Figure 3).
   6. Alternative methods for analyzing GAG disaccharides may also be considered, including chromatography or capillary electrophoresis after fluorescence tagging, fluorophore-assisted carbohydrate electrophoresis (FACE), mass spectrometry, etc. [4, 24–29].

Figure 3.

Example SAX-HPLC spectra A) 8 heparan sulfate (HS) disaccharides commercial standards and B) biological sample digested with heparinase cocktail containing heparinase I, II and III. Peaks marked with an asterisk (*) are non-standard heparan sulfate oligosaccharides [30].

4. Notes

Note 1: The above method for homogenization is for solid tissue that can be pulverized with a mortar and pestle after freezing. Homogenization of other materials (e.g. cells) may require alternative homogenization protocols.

Note 2: Not all samples (e.g. cells) may require delipidation. If samples are low in amount or complexity a simplified procedure may be considered.

Note 3: Lyophilization after the addition of high salt concentrations and sodium borohydride will result in a lot of material following lyophilization. Ensure that the tube used for lyophilization is large enough to handle the volume of the resulting salt material.

Note 4: PD-10 columns utilize Sephadex G-25 size exclusion resins and work in part by volume displacement. These columns are designed for sample volumes up to 2.5 mL. If alternative desalting systems are used make sure to consult the manufacturer’s instructions.

Note 5: Heparinase enzymes can be purchased from a number of vendor sources including New England Biolabs (P0735S, P0736S, P0737S), Sigma (H2519, H6512, H8891), GrampEnz (GE-H0001, GE-H0002, GE-H0003), or IBEX (60–010, 60–018, 60–020). Use a buffer system appropriate to the enzyme according to the manufacturer’s instructions.

Note 6: Disaccharide standards can be purchased either as single disaccharides, or as a complete set of 8 disaccharides commonly found in heparin. Make sure to purchase unsaturated disaccharides.

Note 7: SAX chromatography uses a silica-based quaternary ammonium bonded sorbent and exchanges anions in low to high ionic strength buffers. The columns tend to have wide ranges of pH use (pH 2–8) and are rugged for up to several hundred runs. The column will eventually degrade, which is observed by retention times trending shorter than usual. The retention times of the HS standard mixture can be used to monitor this degradation to determine eventual column replacement.