Proteoglycans - Methods and Protocols Detection of glycosaminoglycans in biological specimens

Abstract

Proteoglycans (PGs) are macromolecules formed by a protein backbone to which one or more glycosaminoglycan (GAG) side chains are covalently attached. Most PGs are present in connective tissues, cell surfaces, and intracellular compartments. The major biological function of PGs derives from the GAG component of the molecule, which is involved in cell growth and proliferation, embryogenesis, maintenance of tissue hydration, and interactions of the cells via receptors. PGs are categorized into four groups based on their cellular and subcellular localization, including cell surfaces and extracellular, intracellular, and pericellular locations. GAGs are a crucial component of PGs involved in various physiological and pathological processes. GAGs also serve as biomarkers of metabolic diseases such as mucopolysaccharidoses and mucolipidoses. Detection of specific GAGs in various biological fluids helps manage various genetic metabolic disorders before it causes irreversible damage to the patient (1). There are several methods for detecting GAGs; this chapter focuses on measuring GAGs using enzyme-linked immunosorbent assay, liquid chromatographic tandem mass spectrometry, and automated high-throughput mass spectrometry.

1. Introduction

Proteoglycans (PGs) are heterogeneous macromolecules consisting of a protein core to which one or more glycosaminoglycan (GAG) chains are covalently attached. PGs structures are diverse in type, size, and composition of polysaccharides attached as various core proteins. GAGs are sulfated negatively charged long linear polysaccharides comprising repeating disaccharide units, uronic acid, and hexosamines. GAGs are classified into five groups, chondroitin sulfate (CS) composed of alternating 1,4-linked β-D-glucuronic acid (GlcA) and 1,3-linked N-acetyl galactosamine (GalNAc) units (2–5), dermatan sulfate (DS) composed of alternating 1,4-linked α-L-iduronic acid (IdoA) and 1,3-linked GalNAc units (2–5), heparin/heparan sulfate (HS) composed of alternating β-1,4-uronic acid (glucuronic/iduronic) and β-1,4-glucosamine (GlcN) units (6–13), keratan sulfate (KS) composed of alternating 3-linked β-D-Galactose (Gal) and 4-linked β-D-GlcNAc units (14, 15) and Hyaluronic acid (HA) consists of repeating β-1,4-D-GlcA and β-1,3-N-GlcNAc units (16, 17). Hyaluronan is an exception in the GAG family since it is a non-sulfated polysaccharide. Based on cellular location, PGs are categorized into four groups: cell surfaces, extracellular, intracellular, and pericellular locations (18). Heparan sulfate proteoglycans (HSPGs) are primarily associated with the plasma membranes of cells and function as major biological modifiers of growth factors such as fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor PDGF (18). These cell surface and pericellular growth factors function as ligand and their cognate receptors in a biologically favorable form. Cell surface PGs are either transmembrane proteins or are covalently attached to glycosylphosphatidylinositol lipid, embedded within the cell membrane. Cell surface PGs are mostly HSPGs or chondroitin sulfate proteoglycans (CSPGs). Iozzo and Schaefer have thoroughly described the classification of PGs and their function (18). Pericellular and basement membrane zone PGs are mostly HSPGs and anchored via integrins or other receptors on cell surfaces; moreover, they can also be a part of most basement membranes. Intracellular PGs are associated with heparin. Extracellular PGs are the largest PG class that includes three classes; one class is associated with hyaluronan. The other class of extracellular PGs is the small leucine-rich proteoglycan (SLRP), which is further categorized into five groups, the first group predominates in CS and DS, the second group with KS, and the third group with DS/CS. Groups four and five are non-canonical and do not have specific GAG components. The third class is associated with calcium-binding HSPGs, which are referred to as secreted protein acidic and rich in cysteine (SPOCK) (18). The major biological function of PGs derives from the GAG component of the molecule, which is involved in cell growth and proliferation, embryogenesis, maintenance of tissue hydration, and interactions of the cells via receptors. GAGs are also biomarkers of certain metabolic disorders such as mucopolysaccharidoses (MPS) and mucolipidoses (ML). Several methods are available to measure GAGs in biological fluids and tissues. However, measurement accuracy is based on certain specific methods such as enzyme-linked immunosorbent (ELISA) assay (19–26), high-performance liquid chromatography (HPLC) coupled with ion exchange chromatography (27), atmospheric-pressure chemical ionization mass spectrometry (28–30), mass spectrometry (31–37), gas spectrometry alone (38–41) or coupled with mass spectrometry (42), capillary electrophoresis alone (43, 44) or coupled with mass spectrometry (45, 46), and automated high-throughput mass spectrometry (47, 48). Dye-specific and thin layer chromatography methods have limited applications and are thus commonly not in practice. This chapter overviews Enzyme-linked Immunosorbent Assay (ELISA), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and automated high-throughput mass spectrometry (HT-MS/MS) methods and protocols for measuring specific GAGs in biological fluids and tissues.

2. Materials

2.1. Enzyme-linked Immunosorbent Assay (ELISA)

ELISA methods were first developed in 1971. The first method used polyclonal antibodies conjugated with alkaline phosphatase (ALP) (49) or horseradish peroxidase (HRP) (50) to capture an antigen of interest. The specific enzyme activity of the conjugate was determined by incubating a solution of known antigen quantity. In 1985, the first ELISA protocol for KS was developed after the characterization of 5-D-4, a KS-specific antibody, in 1983 (51, 52). Our lab used the principle of sandwich assays (in which the wells of an ELISA plate are coated with the capture antibody, and the detection antibody then binds to a different, non-overlapping region of the antigen) (53) to develop an ELISA method to quantify KS in 2004 with the following procedure (20).

2.2. Materials for ELISA

1. All reagents described for the ELISA are commercially available as the KS-ELISA kit KS standard samples.

2. Anti-keratan MAb (5-D-4) purchased from Seikagaku Corp. (Tokyo, Japan).

3. NHS-Biotin.

4. Microtiter plate.

5. Plate washing buffer (PBS-0.05% Tween 20).

6. Sample diluent (PBS containing 1% BSA).

7. 3,3’, 5,5’- tetramethylbenzidine (TMB).

8. The HS, heparin (Hep), CS-A, CS-C, and DS in an acid mucopolysaccharide.

2.2. Liquid chromatography tandem mass spectrometry materials

2.2.1. Standards and Enzymes

1. Unsaturated standard disaccharides, DDiHS-0S [2-acetamido-2-deoxy-4-O-(4-deoxy-a-L-threo-hex-4-enopyranosyluronic acid)-D-glucose], DDiHS-NS [2-deoxy2-sulfamino-4-O-(4-deoxy-a-L-threo-hex-4-enopyranosyluronic acid)-D-glucose], ΔDi-4S [2-acetamido-2-deoxy-4-O-(4-deoxy-a-L-threo-hex-4-enopyranosyluronic acid)-4-O-sulfo-Dglucose], mono-sulfated KS [Galβ1–4GlcNAc(6S)], and di-sulfated KS [Gal(6S)β1–4GlcNAc(6S)].

2. Chondroitinase B, heparitinase, and keratanase II.

3. Chondrosine is used as an internal standard (IS).

4. Stock solutions ΔDiHS-NS (1000 ng/ml), ΔDiHS-0S (1000 ng/ml), ΔDi-4S (1000 ng/ml), mono- and di-sulfated KS (10000 ng/ml) and IS (5 μg/ml) are prepared separately in milliQ water. Standard working solutions of ΔDiHS-NS, ΔDiHS-0S, ΔDi-4S (7.8125, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 ng/ml), and mono- and di sulfated KS (80, 160, 310, 630, 1250, 2500, 5000, and 10000 ng/ml) each mixed with IS solution (5 μg/ml) are prepared (see Note1).

2.2.2. Extraction of GAGs from Tissue

1. Tissue GAGs are isolated as described by Mochizuki et al. (54) with slight modification.

2. Keep the tissue (brain, liver, heart, muscle, spleen, and bone) on dry ice.

3. Dissect and weight 30–50 mg tissues and place them in homogenization tubes (2 ml microtubes pre-filled with 2.8 mm ceramic beads, OMNI international Kennesaw, GA) with 1.0 ml chilled acetone.

4. Homogenize the tissue with Omni Bead Ruptor Bead Mill Homogenizer (OMNI international Kennesaw, GA) with appropriate speed and time (see Note 2).

5. Transfer the tissue homogenate to a 1.5 ml tube and centrifuge for 30 min. at 12,000 × g, at 4°C.

6. Remove acetone buffer and dry pellets completely (using a vacuum centrifuge).

7. Apply 200 μl of 0.5 N NaOH and incubate for 2 h at 50°C.

8. Neutralize by 100 μl of 1 N HCl (see Note 3).

9. Add NaCl powder directly into the solution and make a 3M NaCl sample.

10. Centrifuge for 5 min at 9391 × g, room temperature (RT) and transfer the supernatant to a new tube.

11. Add 83.3 μl of 1 N HCl and centrifuge for 5 min at 9391 × g, RT and transfer the supernatant to a new tube.

12. Neutralize the pH with 83.3 μl of 1 N NaOH (see Note 3).

13. Add 2 times volume (935 μl) of 1.3% potassium acetate in 100% EtOH.

14. Centrifuge samples for 30 minutes at 12,000 × g, 4°C.

15. Remove the solution and add 1 ml of cold 80% EtOH.

16. Invert seral times and centrifuge samples for 10 min. at 12,000 × g, 4°C.

17. Remove the solution and dry pellets completely at RT.

18. Add 100 μl of 50 mM Tris-HCl (pH 7.0).

19. Use 10 μl for pretreatment (described in the method section).

2.2.3. Extraction of GAGs from Dried blood spot (DBS)

1. Two disks (3.3 mm) of DBS spots are obtained by DBS puncher into an AcroPrep^™ Advance 96-Well Filter Plate.

2. 100 μl of 0.1% BSA solution is added to each well, incubated for 15 min, and centrifuged for 15 min at 14.4 × g. Discard the filtrates solution.

3. The pretreatment of DBS is described in the method section.

2.2.4. Apparatus

1. Mass spectrometer:1290 Infinity liquid chromatography system with a 6460 triple quad mass spectrometer (Agilent Technologies, Palo Alto, CA). Agilent Jet Stream Technology is operated with electrospray ionization in the negative ion mode with a drying gas temperature of 350°C, drying gas flow of 11 L/min, nebulizer pressure of 58 PSI, sheath gas temperature 412°C, sheath gas flow of 11 L/min, the capillary voltage of 4000 V and nozzle voltage of 2000 V.

2. Hypercarb column (2.0 mm i.d. 50 mm long; 5 μm particles; Thermo Scientific, USA).

3. The chromatographic system uses a mobile phase: 100 mM ammonia (A) (see Note 4) and 100% acetonitrile (B). The gradient condition is programmed as follows: the initial composition of 100% A is held for 1 min, linearly modified to 30% B to 4 min, maintained at 30% B to 5.5 min, returned to 0% B to 6 min, and maintained at 0% B until 10 min. The flow rate is 0.7 ml/min.

4. Specific precursor and product ions, m/z are used to quantify each disaccharide, respectively (IS, 354.3, 193.1; DS (ΔDi-4S), 458.4, 300.2; mono-sulfated KS, 462, 97; di-sulfated KS, 542, 462; ΔDiHS-NS, 416, 138; ΔDiHS-0S, 378.3, 175.1) [Figure1, near here] (55–58).

5. All other chemicals and solvents are LC-MS/MS grade.

Figure 1.

Chromatogram of 5 GAGs (one DS, 2 HS and two KS) by LC-MS/MS

2.3. Non-reducing end disaccharides materials

2.3.1. Cell lines and culture

1. Human foreskin fibroblasts (HFF): American Type Culture Collection (CRL-1634, Manassas, VA).

2. Human dermal fibroblasts derived from patients (MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS VI, and MPS VII) biopsies: Coriell Institute (Camden, New Jersey).

3. Normal clinically healthy individuals.

4. DMEM: 50 Units/ml penicillin, 50 μg/ml streptomycin, 2 mM glutamine, and 10% fetal bovine serum.

5. Cells grow for 8 weeks to ensure enough GAG for analysis.

6. Measurement of Sulfamidase activity in cell extracts with 4-methylumbelliferyl-α-D-N-sulfoglucosaminide (Moscerdam, The Netherlands).

2.3.2. Urine samples from mice and human

1. MPS IIIA mice (Sgsh−/−): Jackson Laboratory (B6.Cg-Sgsh)

2. Dr. Patricia Dickson (University of California, Harbor) provided canine samples.

3. Dr. Elizabeth Neufeld (University of California, Los Angeles) provided MPS IIIB mice samples.

4. Human urine samples without personal identifying information: Zacharon Pharmaceuticals, Inc. donors from an MPS patient advocacy group with informed consent.

2.3.3. Apparatus

1. A LCQ classic quadrupole ion trap mass spectrometer equipped with an electrospray ionization source and a quaternary high-performance liquid chromatography pump (Thermo-Finnigan, San Jose, CA).

2. Derivatized and non-derivatized disaccharide residues are separated on a C18 reversed-phase column (0.46 × 25 cm, Vydac) with the ion pairing agent dibutylamine.

3. The isocratic steps are: 100% buffer A (8 mM acetic acid, 5 mM DBA) for 10 min, 17% buffer B (70% methanol, 8 mM acetic acid, 5 mM DBA) for 15 min; 32% buffer B for 15 min, 40% buffer B for 15 min, 60% buffer B for 15 min; 100% buffer B for 10 min; and 100% buffer A for 10 min.

4. Ions of interest are monitored in negative ion mode.

5. To minimize in-source fragmentation of sulfated disaccharides, keep the capillary temperature and spray voltage at 140°C and 4.75 kV, respectively.

6. The accumulative extracted ion current (XIC) is computed, and further data analysis is carried out as described in the documentation for the Qual Browser software provided by Thermo-Finnigan.

2.4. Methanolysis materials

2.4.1. Reagents

1. Optima LC-MS grade water.

2. HPLC grade methanol and LC-MS grade acetonitrile (ACN).

3. Dermatan sulfate, chondroitin sulfate A, and heparan sulfate calibration standards.

4. Keratan sulfate calibration standard (Abingdon, UK). Methanolic hydrochloric acid (HCl) 3 N, methanol-d4 99.8% purity, and ammonium acetate 98% purity.

5. Acetyl chloride.

6. Synthetic human urine (Bioreclamation, Hicksville, NY).

2.4.2. Urine collection

• 1.Urine specimens from 58 MPS patients.
• 2.Twelve Hurler, Hurler/Scheie, or Scheie syndromes patients (9 patients treated by bone marrow transplant or ERT).
• 3.Ten Hunter syndrome (6 patients treated by ERT) patients.
• 3.Four Sanfilippo syndrome patients.
• 4.Twenty-five Morquio A syndrome (16 patients treated by ERT) patients.
• 5.Seven Maroteaux-Lamy syndrome (5 patients treated by ERT) patients.
• 6.79 healthy reference control urine specimens.
• 7.Gaucher disease, Fabry disease, alpha-mannosidosis, mucolipidosis II, Pompe disease, GM1 gangliosidosis, aspartylglucosaminuria, and Schindler disease patients urine samples.

2.4.2. Apparatus

1. Xevo TQ-S (Waters Corp., Milford, MA) MS/MS combined with an Acquity I Class (Waters) UPLC system.

2. Electrospray (ESI): in positive ion mode, signals are acquired during a multiple reaction monitoring experiment (MRM).

3. Mass spectrometry acquisition parameters are DS/CS 426, 236; DS IS/CS IS 432, 239; HS 406, 245; HS IS 412, 251; KS 420, 258; KS IS 423, 261.

4. BEH Amide UPLC column (2.1 × 50 mm, 1.7 mm particle size) with an online pre-filter (0.2 mm) is used for chromatographic separation.

5. The method run-time and the total analysis time between injections are 7 and 8 min, respectively—injection volume is 2 μl.

6. Mobile phase A 90:10 ACN:H₂O + 10 mM CH₃COONH₄, Mobile phase B 90:10 H₂O:ACN + 10 mM CH₃COONH₄. Gradient 0.00–1.50 min 0% B, 1.50–2.25 min 0–10% B (linear), 2.25–4.50 min 10% B, 4.50–5.50 min 40% B, 5.50–7.00 min 0% B.

7. Flow rate is 0.3 ml/min.

2.5. Automated high-throughput mass spectrometry materials

2.5.1. Standards and Enzymes

Standards and enzymes are used as mentioned in section 2.2.1

2.5.2. Preparation of DBS for extracting GAGs

DBS punch for GAG extraction was done as described in section 2.2.3

1. For pretreatment of DBS, add a cocktail of 40 μl with recombinant chondroitinase B, heparitinase, and keratanase II (all enzymes, 1 mU/sample) (see Note 5), and IS solution (5 μg/ml) followed by 150 μl of 50 mM Tris- hydrochloric to each well.

2. Place the filter plate on a receiving 96 wells plate and incubate at 37°C overnight.

3. Centrifuge the plate at 14.4 × g for 20 min.

4. Inject the processed samples into RapidFire high-throughput system with 6400 series MS/MS (Agilent Technologies, Inc: Santa Clara, CA).

2.5.3. Establishing interface between Mass spectrometer and RapidFire

1. Turn off engines on Mass spec monitor, turn on configuration, and unselect LC.

2. Turn on Rapidfire software, and open MassHunter QQQ.

3. Open the control panel on the RapidFire monitor, and start the system.

4. Match IP address for mass spec and RapidFire computers.

5. Finally, the connection is established to the program server by clicking ‘Connect’.

2.5.4. Apparatus

1. RapidFire high-throughput system with 400 series MS/MS (Agilent Technologies, Inc: Santa Clara, CA). This injection system interfaces directly with the mass spectrometer. Agilent Jet Stream Technology is operated with electrospray ionization in the negative ion mode with a drying gas temperature of 350°C, drying gas flow of 11 L/min, nebulizer pressure of 40 PSI, sheath gas temperature 200°C, sheath gas flow of 11 L/min, the capillary voltage of 4000 V and nozzle voltage of 500 V.

2. The RapidFire microscale solid-phase extraction cartridge D (graphitic carbon) aspirates 10 μl of the sample per well and desalts it with HPLC grade water in a 2.75-sec wash cycle. The pump flow rate is fixed based on the best fit (see Note 6).

3. Analytes are then coeluted into MS in a 0.6-sec elution cycle with 25% acetone, 25% acetonitrile, and 100 mM ammonia.

4. MassHunter QQQ mass spectrometer in negative ESI mode is utilized to measure specific precursor, product ions, and m/z, following multiple-reaction monitoring transitions measured for each disaccharide, respectively for IS, at 354.3, 193.1; DS (ΔDi-4S), 458.4, 300.2; mono-sulfated KS, 462, 97; di-sulfated KS, 542, 462; ΔDiHS-NS, 416, 138; ΔDiHS-0S, 378.3, 175.1 (55–58).

5. Integrate the sample data using the RapidFire Integrator software (Agilent Technologies, Inc: Santa Clara, CA), resulting in individual AUC (area under the curve) values for each analyte in each sample.

3. Methods

3.1. ELISA

3.1.1. Sandwich ELISA Assay to Quantify KS

• 2.Precoat plates with antibodies by adding 50 μl of 20 μg/ml antibody solution to each well of the microtiter plate.
• 3.Biotinylate 5-D-4 antibody using NHS-Biotin.
• 4.Bring plate washing buffer and sample diluent to room temperature before use.
• 5.Add 200 μl/well of washing buffer to the microplate. Then discard the washing buffer.
• 6.Repeat the washing procedure three times.
• 7.Add 50 μl/well of diluted unknown samples or KS standards to the microplate.
• 8.Incubate at 37°C for 60 min.
• 9.Perform washing procedure four more times.
• 10.Add 25 μl/well of horseradish peroxidase-conjugated streptavidin and 25 μl/well of the biotinylated antibody.
• 11.Incubate at 37°C for 60 min.
• 12.Wash the plate four more times.
• 13.Add 50 μl/well of substrate solution (TMB).
• 14.Incubate at room temperature for 10 min.
• 15.Stop the reaction with 50 μl 1 N HCl.
• 16.Measure the absorbance at 450 nm with a microplate spectrophotometer.
• 17.Read KS concentration by applying the absorbances of each sample to the calibration curve.

3.1.2. Sandwich ELISA Assay to Quantify HS

Our lab developed a similar sandwich assay to quantify HS in 2005 (25). This method followed the same procedure as the KS sandwich assay but used a monoclonal antibody raised against HS.

3.2. Liquid chromatography tandem mass spectrometry

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a novel technique with numerous applications in detecting various analytes. The principle of mass spectrometry is to detect compounds by mass to charge ratio (m/z), developed after the discovery of the electron by John Thomson in 1897 (59). Mass spectrometry is a sensitive, specific, and accurate method for GAG analysis. Several protocols have been developed in the past for the measurement of GAGs in blood (plasma/serum), urine, cerebrospinal fluid (CSF), and DBS, which are summarized below.

3.2.1. Enzyme-mediated degradation of polysaccharides to disaccharides and detection by LC-MS/MS

Oguma et al. applied the detection strategy of macromolecule GAGs by utilizing enzymes to digest polysaccharides to disaccharides. They developed a method to detect HS in mouse tissues using the heparitinase enzyme and measured by API-4000 mass spectrometer equipped with a Turbo Ion Spray in negative ion mode using Hypercarb (2.0 mm i.d. × 150 mm, 5 μm) column (35). Using the keratinase II enzyme, they have developed mono-sulfated and di-sulfated KS detection from bovine and rodent tissues (36). Subsequently, they validated CS/DS in mice tissue using chondroitin B enzyme and measured by LC-MSMS (37). Later, Oguma et al. developed protocols to determine KS, DS, and HS from human serum/plasma using a similar approach described above (33, 34). The enzymatic (chondroitinase ABC, hyaluronidase, heparitinase, and keratanase) digestion of polysaccharides was also adapted by Osago et al. (60); however, they developed a better method using LC-MS/MS to quantify 23 various sulfated GAGs (8 CS/DS, 1 hyaluronic acid, 12 HS, and 2 KS) simultaneous with a selected reaction monitoring (SRM) in negative ESI mode. Their method quantifies simultaneously internal and non-reducing terminal saccharides that were useful for estimating the chain length of GAGs. Recently, Arunkumar et al. used five plex assay measuring five lysosomal enzymes (α-L-iduronidase (MPS I), iduronate-2-sulfatase (MPS II), α-N-acetylglucosaminidase (MPS IIIB), N-acetylglucosamine-6-sulfatase (MPS IVA), and N-acetylglucosamine-4-sulfatase (MPS VI)) and five GAGs (two kinds of HS, DS, and two kinds of KS) in DBS to diagnose suspected MPS patients using LC-MS/MS (61).

We adapted Oguma et al. method with slight modification by utilizing enzyme-mediated degradation of polysaccharides to disaccharides, DS, KS, and HS by using chondroitinase B, keratinase II, and heparitinase, respectively [Figure 2, near here]. The detailed method is described below (55–58).

Figure 2:

Production of Disaccharides after enzymatic digestion of GAGs. KS: Keratan sulfate; HS: heparan sulfate; DS: dermatan sulfate; CS: chondroitin sulfate.

1. Add 10 μl of each plasma/serum, urine sample, or standard and 90 μl of 50 mM Tris– hydrochloric acid buffer (pH 7.0) in wells of AcroPrep^™ Advance 96-Well Filter Plates that have Ultrafiltration Omega 10 K membrane filters (PALL Corporation, NY, USA).

2. Add a cocktail of 40 μl with recombinant chondroitinase B, heparitinase, and keratanase II (all enzymes, 1 mU/sample), and IS solution (5 μg/ml) followed by 60 μl of 50 mM Tris- hydrochloric acid buffer to each well.

3. Place the filter plate on a receiving 96 wells plate and incubate at 37°C overnight.

4. Centrifuge the plate at 14.4 × g for 20 min.

5. Inject the processed samples into LC-MS/MS.

6. Tissues and DBS GAGs are also analyzed as above (1–5).

7. The levels of GAGs in urine samples are normalized by creatinine, measured with a Creatinine (urinary) Colorimetric Assay Kit (19, 21, 55, 62, 63).

3.2.2. Non-reducing ends disaccharides measurement by LC-MS/MS

Lawrence et al. developed a method to detect the non-reducing end of GAGs (64, 65). In this method, the lack of lysosomal enzymes results in the accumulation of characteristic non-reducing terminal carbohydrate structures. GAGs are enzymatically depolymerized, releasing unique mono-, di-, or trisaccharides from the non-reducing ends of the chains, which are subsequently labeled by reductive amination with heavy isotope-labeled aniline. The modified sugars are quantified by LC-MS/MS.

1. Digest cell lysates and urine HS and CS (1–10 pmoles) with enzymes and dry in a centrifugal evaporator.

2. Add [12C6]aniline or [13C6]aniline (15 μl, 165 μmol) and 15 μl of 1 M NaCNBH₃ freshly prepared in dimethylsulfoxide: acetic acid (7:3, v/v) to each sample.

3. Reactions are carried out at 65°C for 4 h or 37°C for 16 h, dry in a centrifugal evaporator.

4. Unsubstituted amines are reacted with propionic anhydride. Reconstitute the dried samples in 20 μl of 50% methanol and 3 μl of propionic anhydride (23.3 μmol). Reactions are carried out at room temperature for 2 h.

5. Acylated disaccharides are subsequently aniline-tagged, as described above.

6. Each sample is mixed with commercially available standard unsaturated disaccharides, standard N-sulfoglucosamine, glucosamine-6-sulfate, N-acetylgalactosamine-4-sulfate, and N-acetylgalactosamine-6-sulfate, and/or β-D-idopyranosyluronate)-(1→4)-(2-N-acetyl-2-deoxy-α/β-D-glucopyranoside that is synthesized.

7. All standards are tagged with [13C6]aniline.

8. Samples are then analyzed by liquid chromatography-mass spectrometry using an LTQ Orbitrap Discovery electrospray ionization mass spectrometer (Thermo Scientific) equipped with a quaternary high-performance liquid chromatography pump (Finnigan Surveyor MS pump) and a reverse-phase capillary column.

This method can clearly distinguish 8 different forms of MPS from unaffected controls. This sophisticated derivatization method has not yet been adapted for higher throughput methods needed for routine laboratory use or newborn screening (NBS) but shows promise as a method to identify very selective biomarkers.

3.2.3. Methanolysis

Auray-Blais et al. developed the protocol, which uses chemical degradation of polysaccharides (66–68). After methanolysis, the liberated disaccharides from the urine of MPS patients are measured by LC-MS/MS (69).

1. Twenty-five microliters of homogenized urine samples are deposited in a disposable borosilicate glass culture tube fitted with a screwcap (16 × 100 mm).

2. After evaporation of the urine sample under a stream of nitrogen, 500 μl of a commercial methanolic HCl 3 N solution is added.

3. Cap the tubes, vortex, and incubate at 65°C for 1 h.

4. After incubation, samples are immediately evaporated under a stream of nitrogen, then suspended in 200 μl of the resuspension solution.

5. Samples are transferred to ultra-performance liquid chromatography (UPLC) vials and centrifuged prior to the injection of 2 μl in the UPLC-MS/MS system.

6. Cerebrospinal fluid (CSF) (70, 71) and animal tissues (72) are also done with this method.

Trim et al. applied butanolysis derivatization followed by LC-MS/MS to quantify HS in urine from MPS patients. (MPS I, II, III, and VI) (73). Most recently, Forni et al. reported simultaneously quantifying urinary HS and DS from MPS (I, II, III, VI, and VII) patients using butanolysis derivatization followed by LC-MS/MS (74).

3.2.4. Automated high-throughput mass spectrometry (HT-MS/MS)

The automated high-throughput mass spectrometry (HT-MS/MS) or Rapidfire system is a very fast autosampler that eliminates the bottleneck of throughput, simultaneously maintaining the reliability and quality of the standard LC-MS/MS-based methods. RF-MS/MS excludes chromatographic separation, reducing sample-to-sample cycle times to seconds, thus allowing sample processing within seconds (7–13 sec/sample) and the possibility of analysis of over a million samples annually (75). Originally, this method was used for amino acid and acylcarnitine profiling to diagnose inborn errors of metabolism disorders in newborn DBS (47). The underlying method involves sample aspiration into a solid-phase extraction cartridge for desalting and concentration, following direct injection into MS/MS without chromatographic separation, which makes this system yield faster throughput than standard LC-MS/MS-based methods (48). The RF-MS/MS system is highly flexible in adapting to different settings, as was validated in a study where a novel BLAZE mode was adapted instead of standard batch mode to develop a fast cartridge-free system, which improved sensitivity, peak shape, and reduced time to less than 3 sec/sample (76). The RF-MS/MS system has been validated to be suitable for applications in various drug discoveries for profiling multiple compounds (48, 56, 77, 78) and ADME (Absorption, Distribution, Metabolism, and Excretions) properties (48, 56, 76). Tomatsu et al. (75) developed a highly sensitive and accurate LC-MS/MS and RF-MS/MS method for four specific GAGs detection HS, CS, KS, and DS by digestion of the purified polymer CS (shark cartilage), DS (pig skin), HS (bovine kidney), and KS (bovine cornea) by a mixture of chondroitinase C, chondroitinase B, heparitinase, and keratanase II.

1. Centrifuge the samples to be measured to remove insoluble material.

2. Mix 10 μl of the supernatant with 90 μl of Tris-HCL Buffer (pH 7.0) in a filter well plate. Centrifuge the filter well plate with receiving 96 well plate at 14.4 × g for 15 min to remove low molecular compounds.

3. Add 20 μl of internal standard (500 ng/ml chondrosine), 20 μl of 50 mM Tris-HCl buffer (pH 7.0), and 10 μl of chondroitinase C, chondroitinase B, heparitinase, and keratanase II, respectively, (each 2 mU/10 μl of 50 mM Tris-HCl buffer) to each well of filter plate.

4. Incubate filter well plates with samples shaking at 37°C for 15 h to digest the GAGs.

5. Centrifuge filter plate with new receiving 96 well plate at 14.4 × g for 15 min.

6. Add 20 μl of ddH₂O to each flowthrough sample and mix by vortexing for 10 sec.

7. Analyzed the samples using the RapidFire high-throughput mass spectrometry platform.

The results validated that the RF-MS/MS system has a 25–100-fold faster throughput than the traditional LC-MS/MS method and deemed it suitable for mass screening applications, like newborn screening (75). Shimada et al. utilized HT-MS/MS system to develop a higher throughput system to assay heparan sulfate levels in blood samples from various MPS patients and control subjects and dried blood spots from newborns controls and determined that HT-MS/MS has comparable sensitivity and specificity to conventional LC-MS/MS-based methods, making it suitable for diagnosis, monitoring, and newborn screening of various types of MPS (48).

We have developed a method with slight modification by comparing different organic solutions and compatible matrix, flow rates, parameters such as temperature, voltage, and utilizing enzyme-mediated degradation of polysaccharides to disaccharides, DS, KS, and HS by using chondroitinase B, keratinase II, and heparitinase, respectively (Figure 2). The detailed method is described below.

1. Take two 3.00 mm disks of DBS spots by DBS puncher into an AcroPrep^™ Advance 96-Well Filter Plate.

2. Treat the disks with 100 μl of 0.1% BSA solution, incubate for 15 min, and centrifuge at 14.4 × g for 15 min. Discard the filtrate solution.

3. For the pretreatment of DBS, add a cocktail of 40 μl with recombinant chondroitinase B, heparitinase, and keratanase II (all enzymes, 1 mU/sample), and IS solution (5 μg/ml) to each well, followed by the addition of 150 μl of 50 mM Tris- hydrochloric acid to each well.

4. Keep the filter plate on a receiving 96 wells plate and incubate at 37°C overnight, followed by centrifugation at 14.4 × g for 20 min.

5. Inject the processed samples into RapidFire high-throughput system with 400 series MS/MS.

We have analyzed 23 controls, 4 MPS I, 13 MPS II, and 2 MPS IIIB DBS samples by RapidFire (Table 1). Three GAGs (two HS, one KS) and IS peaks of RapidFire are presented [Figure 3, near here]. The overall sample analysis by LC-MS/MS and RapidFire is presented [Figure 4, near here]. There are several advantages and disadvantages of these protocols, which are summarized in Table 2.

Table 1.

Analysis of control and MPS DBS samples by RapidFire

Sample Type	Sample Number	Di HS 0S (ng/ml)	Di HS NS (ng/ml)	KS (ng/ml)
Control	23	174 ± 63	248 ± 125	705 ± 483
MPS I	4	186 ± 88	349 ± 161	1015 ± 339
MPS II	13	300 ± 159	239 ± 83	782 ± 221
MPS IIIB	2	444 ± 98	379 ± 142	707 ± 360

Figure 3.

Standards of 3 GAGs with serial dilution (2HS, 1KS) and IS peaks for these standards by RapidFire

Figure 4.

Schematic presentation of sample analysis by LC-MS/MS and RapidFire

Table 2.

Advantages and limitations of various methods for GAGs analysis

Methods	Advantages	Drawback	References
ELISA	Measurement of HS, KS, CS and DS in blood and urine samples	Can not detect multiple GAGs simultaneously. It is also costly.	(20, 25, 26, 79, 80)
Enzymatic degradation and LC-MS/MS	Simultaneous Measurement of HS, KS, CS and DS in plasma, serum, urine, DBS, tissue extracts, and CSF	Variation in the ionization efficiency of different disaccharides. Enzymes are also costly.	(33, 37, 55, 58, 60, 81)
Non-reducing end of GAG and LC-MS/MS	Measurement of HS, DS in blood urine, and tissues	This method is not applicable to identify KS in blood or urine	(65)
Methanolysis and LC-MS/MS	Measurement of CS, DS, HS, in urine, blood, CSF, and tissues. KS in urine only.	Not applicable for blood KS. No detection of subclass of CS, HS, and KS	(66, 68, 70, 71)
Automated high throughput MS/MS (RapidFire)	Measurement of HS, KS, and DS in blood and DBS within 7 seconds per sample.	Isomeric disaccharides cannot be distinguished	(48, 63)

4. Notes

1. Take the stocks as follows in order to make the highest standard name as standard 8

   Mono-sulfated KS	10 μl of stock
   Di-sulfated KS	10 μl of stock
   Di-HS-0S	10 μl of stock
   Di-HS-6S	10 μl of stock
   Di-HS-NS	10 μl of stock
   Di-6S	4 μl of stock
   Di-4S	4 μl of stock
   H2O	942 μl

   Do serial dilution to make standards 7 through standar1 as following;

   STD 7	STD 6	STD 5	STD 4	STD 3	STD 2	STD 1
   Std 8 (500 μl)	Std 7 (500 μl)	Std 6 (500 μl)	Std 5 (500 μl)	Std 4 (500 μl)	Std 3 (500 μl)	Std 2 (500 μl)
   Add 500 μl water	Add 500 μl water	Add 500 μl water	Add 500 μl water	Add 500 μl water	Add 500 μl water	Add 500 μl water

2. For soft tissues such as liver, brain, spleen and heart, use speed 6 for 20 sec. For trachea and bone use speed 8 for 30 sec. to make sure the tissues are completely homogenized.

3. Neutralizing with acid or base requires additional diluted acid or base to make sure pH is 7.0, using pH strips.

4. Ammonia concentration in ammonium hydroxide solution is14.8 M. To make 100 mM ammonia take 6.757 ml ammonium hydroxide in 1L of milliQ water.

5. Recombinant chondroitinase B, heparitinase, and keratanase II each enzyme is 100 mU lyophilized powder. Reconstitute each enzyme with 1% BSA and take 10 μl each for one sample (1mU/sample). Stock internal standard (IS) is 1 mg/ml. Take 3 μl in 600 μl milliQ water. Thus in 40 μl cocktail, 10 μl of each three enzymes and 10 μl IS, add to each well.

6. Establishing pump flow rate is crucial for reproducing LC-MS/MS signals. Check the backpressure (lower than recommended pressure 1 bar) or overpressure (generally due to a clog) of the pumps and adjust the flow rate if the backpressure is low. Once the optimum flow rate is identified, the signal on LC-MS/MS will be sharp, and the pressure will be stable.