Apr 28, 2026
Sample preparation of label-free GAG disaccharides from biological tissues
- H Davies-Strickleton1,2,
- James Allsey2,
- Douglas Dyer3,
- David Knight2
- 1Discovery Research Platform, Manchester Cell-Matrix Centre, University of Manchester, UK;
- 2Biological Mass Spectrometry Core Facility, University of Manchester, UK;
- 3Manchester Cell-Matrix Centre, Lydia Becker Institute of Immunology and Inflammation, University of Manchester, UK
- BioMS CRF, UoM
Protocol Citation: H Davies-Strickleton, James Allsey, Douglas Dyer, David Knight 2026. Sample preparation of label-free GAG disaccharides from biological tissues . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg31yxel25/v1
Manuscript citation:
A manuscript of this work is in preparation, which details the method and demonstration of its utility by application to a range of different samples as biological sources.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: October 09, 2025
Last Modified: April 28, 2026
Protocol Integer ID: 229392
Keywords: Glycosaminoglycans, Heparan sulphate, Chondroitin sulphate, Dermatan sulphate, Hyaluronan, Sample preparation, GAG disaccharides, gag disaccharide preparation sample preparation of label, gag disaccharide preparation sample preparation, free gag disaccharides from biological tissue, gag disaccharide preparation, gag disaccharide standard, free gag disaccharide hilic, gag disaccharide, free gag disaccharide, hs disaccharide, disaccharide, free glycosaminoglycan, ha disaccharide, ms analysis from biological tissue, sample preparation of label, digestion with heparinase, biological tissues equimolar stock solution preparation, dnase digestion, sample preparation, ms data acquisition, other biomolecules by pronase
Funders Acknowledgements:
Wellcome Trust Discovery Research Platform (HDS, DD)
Grant ID: 226804/Z/22/Z
Wellcome Trust Career Development Award (DD)
Grant ID: 319823/Z/24/Z
Abstract
This protocol provides a detailed step-by-step guide to generate label-free glycosaminoglycan (GAG) disaccharides from diverse biological materials. The steps involve liberation of GAGs from other biomolecules by pronase and DNase digestion, enrichment of full-length GAGs by centrifugation through molecular weight cut off (MWCO) filters, digestion with heparinases I, II and III (for HS disaccharides) or chondroitinase ABC (for CS/DS/HA disaccharides), and clean up.
This is the second protocol in a collection, which documents the details of all steps required for label-free GAG disaccharide HILIC-MS/MS analysis from biological tissues:
- Sample preparation of label-free GAG disaccharides from biological tissues
Following sample disruption in protocol 1, the GAG disaccharides generated here can then be vialled and analysed according to protocols 4 and 5 in the collection.
Materials
The reagents listed are those used in the development of this protocol. Some reagents are also available from alternative suppliers.
Protocol materials
Ammonium acetateVWR International (Avantor)Catalog #84885.180
Ammonia solution 25% for HPLMerck MilliporeSigma (Sigma-Aldrich)Catalog #5438300100
Calcium chlorideMerck Millipore (EMD Millipore)Catalog #102391
Magnesium chloride hexahydrateFischer ScientificCatalog #10647032
PronaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #10165921001
DNAse IRocheCatalog #10104159001
Bacteroides Heparinase I - 600 unitsNew England BiolabsCatalog #P0735L
Bacteroides Heparinase II - 200 unitsNew England BiolabsCatalog #P0736L
Bacteroides Heparinase III - 35 unitsNew England BiolabsCatalog #P0737L
Chondroitinase ABC from Proteus vulgarisMerck MilliporeSigma (Sigma-Aldrich)Catalog #C2905-10UN
∆UA-2S GlcNCOEt-6S (I-P) internal standard disaccharideIduronCatalog #HD009
Before start
Experimental design
Consider discussing experimental design with the BioMS core facility prior to commencing this protocol. As a minimum include a suitable biological Positive Control Sample to put through the protocol (e.g. lung tissue homogenate) to track the consistency of GAG disaccharide amount, total GAG amount and disaccharide composition.
Biological safety
Handling of biological materials requires knowledge of the risk of infection and this information should be assessed before starting this protocol.
- Consider handling samples in biological safety cabinets to protect the user from any risk of infectious agents
- Pronase treatment and heat deactivation at 98 oC is considered sufficient to render the majority of Group 2 biological agents deactivated, however, advice should be sought based on the risks of each sample type.
Stock preparation
Note
The following stocks, unless stated otherwise, can be prepared and stored at 4 oC for up to one year.
5 M Ammonium Acetate Stock, pH 7
Prepare around 50 mL, based on the following example:
- Weigh ~19.25 g Ammonium acetateVWR International (Avantor)Catalog #84885.180
- Add the required volume of ddH2O using the formula below (for 19.25 g, add 50 mL ddH2O)
- Adjust to 7 by addition of a small volume of Ammonia solution 25% for HPLMerck MilliporeSigma (Sigma-Aldrich)Catalog #5438300100
Note
Exact weights and volumes can be adjusted using the formula below.
Weigh approximately 19.25 g ammonium acetate, then use the formula below to calculate the volume of ddH2O to add (V)
V = m / (C*MW)
where:
V = desired volume (L)
m = mass of solute (g)
C = desired concentration (M; mol/L) = 5
MW = molecular weight (g/mol) = 77.08
Example
If 20 g ammonium acetate was weighed then the volume required would be:
20/(5*77.08) = 0.05189 L or 51.89 mL
300 mM Calcium Chloride Stock
Prepare around 50 mL, based on the following example:
- Dissolve 1.665 g
Calcium chlorideMerck Millipore (EMD Millipore)Catalog #102391 in 50 mL ddH2O
Note
Exact weights and volumes can be adjusted using the formula below.
Weigh approximately 1.665 g CaCl2, then use the formula below to calculate the volume of ddH2O to add (V)
V = m / (C*MW)
where:
V = desired volume (L)
m = mass of solute (g)
C = desired concentration (M) = 0.3
MW = molecular weight = 110.98
Example
If 2g CaCl2 was weighed then the volume required would be:
2/(0.3*110.98) = 0.0600 L or 60 mL
5 mM Magnesium Chloride Stock
Prepare around 50 mL, based on the following example:
- Dissolve 50.8 mg Magnesium chloride hexahydrateFischer ScientificCatalog #10647032
- Add 50 mL ddH2O
Note
Exact weights and volumes can be adjusted using the formula below.
Weigh approximately 50.8 mg (0.0508 g) MgCl2, then use the formula below to calculate the volume of ddH2O to add (V)
V = m / (C*MW)
where:
V = desired volume (L)
m = mass of solute (g)
C = desired concentration (M) = 0.005
MW = molecular weight = 203.3
Example
If 100 mg MgCl2 was weighed then the volume required would be:
0.10/(0.005*203.3) = 0.09837 L or 98.37 mL
Buffer and solution preparation
500 mM Ammonium Acetate Solution, pH 7
Dilute 5 M Ammonium Acetate 1 in 10. For example to make 50 mL:
- Add 45 mL ddH2O
- Add 5 mL 5 M Ammonium Acetate Stock pH 7 for stock
- Mix well, check pH, adjust to 7 if needed
- Can be stored for 6 months at 4 oC
100
Dilute 300 mM Calcium Chloride Stock 1 in 3000. For example to make 15 mL:
- Add 15 mL ddH2O
- Add 5 µL 300 mM Calcium Chloride Stock for stock
- Mix well
- Can be stored for 6 months at 4 oC
5 μM Magnesium Chloride Solution
Dilute 5 mM Magnesium Chloride Stock 1 in 1000:
- Add 9.990 mL ddH2O
- Add 10 µL 5 mM Magnesium Chloride Stock for stock
- Mix well
- Can be stored for 3 months at 4 oC
10 x Heparinase Buffer
Note
Composition of 10 x Heparinase Buffer:
- 400 mM Ammonium Acetate, pH 7 (1 in 12.5 dilution of the 5 M Ammonium Acetate stock)
- 30 mM Calcium Chloride (1 in 10 dilution of the 300 mM Calcium Chloride stock)
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The following formula was used to calculate the volume of each component required to prepare 10 x Heparinase Buffer:
rearranged provides:
where:
C1 = Starting concentration (mM)
V1 = Volume of stock needed ( μL)
C2 = Desired concentration (mM)
V2 = Desired total volume (μL)
e.g. The volume of 300 mM Calcium Chloride required to prepare 50 mL of 10x Heparinase Buffer with a final concentration of 30 mM is:
- V1 = (30*50000)/300 = 5000 μL
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Below, the preparation of 50 mL 10 x Heparinase Buffer is described.
- Should a higher volume be needed (e.g. to prepare 100 mL), use the formula above or simply multiply all component volumes accordingly (e.g. for total volume 100 mL, multiply all the volumes by 2).
Example preparation of 50 mL 10x Heparinase Buffer:
- Add 41 mL ddH2O
- Add 4000 µL 5 M Ammonium Acetate Stock for stock
- Add 5000 µL 300 mM Calcium Chloride Stock (1 in 10) for stock
- Mix well
- Check pH, adjust to 7 if needed
- Can be stored for 6 months at 4 oC
10 x Chondroitinase Buffer
Note
Composition of 10 x Chondroitinase Buffer:
- 400 mM Ammonium Acetate, pH 7 (1 in 12.5 dilution of the 5 M Ammonium Acetate stock)
- 30 μM Calcium Chloride (Note this is 1000 fold lower than the concentration in the 10x Heparinase Buffer; 1 in 10000 dilution of the 300 mM Calcium Chloride Stock. )
-----------------------------------------------------------------------------------------------------
The following formula was used to calculate the volume of each component required to prepare 10 x Chondroitinase Buffer:
rearranged provides:
where:
C1 = Starting concentration (mM)
V1 = Volume of stock needed ( μL)
C2 = Desired concentration (mM)
V2 = Desired total volume (μL)
e.g. The volume of 300 mM Calcium Chloride required to prepare 50 mL of 10x Chondroitinase Buffer with a final concentration of 30 μM is:
- V1 = (0.03*50000)/300 = 5 μL
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Below, the preparation of 50 mL 10 x Chondroitinase Buffer is described.
- Should a higher volume be needed (e.g. to prepare 100 mL), use the formula above or simply multiply all component volumes accordingly (e.g. for total volume 100 mL, multiply all the volumes by 2).
Example preparation of 50 mL 10 x Chondroitinase Buffer:
- Add 46 mL ddH2O
- Add 4000 µL 5 M Ammonium Acetate Stock for stock
- Add 5 µL 300 mM Calcium Chloride Stock (1 in 10000) for stock preparation
- Mix well
- Check pH, adjust to 7 if needed
- Can be stored for 6 months at 4 oC
Pronase Buffer
Note
Composition of Pronase Buffer:
- 50 mM Ammonium Acetate pH 7 (1 in 10 dilution of the 500 mM Ammonium Acetate solution)
- 1
μM Calcium Chloride (1 in 100 dilution of the 100 μM Calcium Chloride Solution)
Prepare fresh stock monthly.
Ensure you have enough Pronase Buffer for all samples
- 0.2 mL is required per sample
- Therefore, 24 mL in the example below will be sufficient for > 100 samples
- For higher numbers of samples, multiply the volumes below accordingly (eg for 200 samples, multiply by a factor of 2)
---------------------------------------------------------------------------------------------------
The following formula was used to calculate the volume of each component required to prepare Pronase Buffer:
rearranged provides:
where:
C1 = Starting concentration (mM)
V1 = Volume of stock needed ( μL)
C2 = Desired concentration (mM)
V2 = Desired total volume (μL)
e.g. The volume of 100 μM Calcium Chloride required to prepare 24 mL of Pronase Buffer with a final concentration of 1 μM is:
- V1 = (0.001*24000)/0.1 = 240 μL
Example preparation of 24 mL Pronase Buffer:
- Add 21.36 mL ddH2O
- Add 2.4 mL 500 mM Ammonium Acetate Stock pH 7 for solution
- Add 240 µL 100 μM Calcium Chloride Solution for solution
- Mix well
- Can be stored for 1 month at 4 oC
Sample tubes
Put all samples in 1.5 mL microcentrifuge tubes, label the tubes.
Note
Here, this disrupted material is used to prepare label-free GAG disaccharides.
All samples for testing are referred to as Test Samples.
In addition to this, a Positive Control Sample (e.g. lung tissue homogenate) should be prepared alongside the Test Samples to be analysed. This serves to assess the consistency of this preparation of samples for GAG disaccharide analysis with previous analyses.
Pronase digestion
Prepare fresh 1 mg/mL Pronase Stock
Note
Prepare immediately before use.
Open and handle Pronase inside a fume hood to prevent inhalation.
Make a single Pronase Stock for all samples (plus 20% extra for spillage).
-------------------------------------------------------------------------------------------------
Calculate the volume of Pronase Stock required
Each sample requires 0.2 mL 1 mg/mL Pronase stock (this is deemed sufficient for the amount of samples outlined in the sample disruption protocol).
Calculate the volume of Pronase Stock required (including 20 % extra for spillage):
Vpronasestock (mL) = 1.2 * (Vper sample (mL) * Nofsamples)
where:
- Vper sample = 0.2 mL
Example volume of Pronase Stock required for 50 samples:
- 1.2 x (0.2 mL x 50)= 12 mL
Calculate the amount of Pronase to be weighed to give 1 mg/mL at the required volume
Calculate the mass of Pronase required:
Mass of Pronase (mg) = C * V
where:
C = 1 mg/mL
V = calculated in the step above (here 12 mL)
Example mass of Pronase required for 12 mL Pronase Stock (for 50 samples):
Mass of Pronase (mg) = (1 * 12) = 12 mg
Weigh the required amount of Pronase, and prepare Pronase Stock:
For example, for 50 samples prepare 12 mL 1 mg/mL Pronase Stock (see above for calculations):
- Weigh 12 mg PronaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #10165921001
- Add 12 mL Pronase Buffer for buffer preparation
- Mix well
Note
Exact weights and volumes can be adjusted using the formula below.
Weigh approximately 12 mg Pronase, then use the formula below to calculate the volume of Pronase Buffer to add (V)
V = m / C
where:
V = volume (mL)
m = mass of solute (mg)
C = concentration (mg/mL) = 1
Example
If 14 mg Pronase was weighed then the volume required would be:
14/1 = 14 mL
Add 200 µL 1 mg/mL Pronase Stock to each sample and mix briefly
Incubate at 37 °C overnight (mixing not required)
Vortex samples and continue incubation for 1 hour
Heat deactivate at 98 °C for 10 minutes
DNase I digestion
Prepare 4 mg/mL DNase I Stock
Note
Prepare immediately before use.
Open and handle DNase inside a fume hood to prevent inhalation.
Make a single DNase I Stock for all samples (plus 20 % extra for spillage).
-------------------------------------------------------------------------------------------------
Calculate the volume of DNase I Stock required
Each sample requires 20 μL 4 mg/mL DNase stock (this is deemed sufficient for the amount of samples outlined in the sample disruption protocol).
Calculate the volume of DNase I Stock required (including 20 % extra for spillage):
Vdnasestock (mL) = 1.2 * (Vper sample (mL) * Nofsamples)
where:
- Vper sample = 0.02 mL
Example volume of DNase I Stock required for 50 samples:
- 1.2 x (0.02 mL x 50) = 1.2 mL
Calculate the amount of DNase I to be weighed to give 4 mg/mL at the required volume
Calculate the mass of DNase I required:
Mass of DNase I (mg) = C * V
where:
C = 4 mg/mL
V = calculated in the step above (here 1.2 mL)
Example mass of DNase I required for 1.2 mL DNase I Stock (for 50 samples):
Mass of DNase I (mg) = (4 * 1.2) = 4.8 mg
Weigh the required amount of DNase I, and prepare DNase I Stock:
For example for 50 samples prepare 1.2 mL 4 mg/mL DNase I Stock:
- Weigh 4.8 mg DNAse IRocheCatalog #10104159001
- Add 1.2 mL 5 μM Magnesium Chloride Solution for solution preparation
- Mix well
Note
Exact weights and volumes can be adjusted using the formula below.
Weigh approximately 5 mg DNase I, then use the formula below to calculate the volume of 5 μM Magnesium Chloride Solution to add (V)
V = m / C
where:
V = volume (mL)
m = mass of solute (mg)
C = concentration (mg/mL) = 4
Example
If 6 mg DNase I was weighed then the volume required would be:
6/4= 1.5 mL
Add 20 µL 4 mg/mL DNase I stock to each sample
Mix by vortexing briefly
Incubate at 37 °C for 1 hour (mixing not required)
Note
Samples can be frozen here if needed.
GAG enrichment by molecular weight cut-off (MWCO) filtration
Thaw samples if required
Centrifuge samples at 4 °C for 10 minutes at 15000 rpm
Pre-wash 500 μL 3 kDa MWCO filters with 200 µL ddH2O by centrifugation at 14000 x g for 10 minutes (discard flow through)
Equipment
Amicon Ultra-0.5 Centrifugal Filter Unit
NAME
Centrifugal Filter Unit
TYPE
Millipore
BRAND
UFC5003BK
SKU
LINK
Load sample supernatants into pre-washed MWCO filters
Centrifuge MWCO filters at14000 x g for 25 minutes at ambient temperature, or until the retentate volume is 50-100 µL
- Discard the flow through
Wash retentate with 400 µL ddH2O by centrifugation at 14000 x g for 25 minutes, or until 50-100 µL remains
- Discard the flow through
Repeat the wash step and spin until the volume of the retentate is 20-50 µL
Invert the MWCO filter and place inside a fresh tube to collect the sample by centrifugation at1000 x g for 2 minutes
Pipette up/down and transfer samples to 1.5 mL protein lobind tubes
- Label the lids of the tubes (these will be re-used later)
OPTIONAL: If samples are to be analysed for both HS and CS/DS/HA disaccharides then divide the sample into two aliquots, one for HS analysis and for for CS/DS/HA analysis.
The amount of sample to be used for HS and CS/DS/HA may vary (for instance some tissues have much higher CS/DS compared to HS).
In the first instance divide the sample into two equal volumes:
- Use one half for HS preparation and the other half for CS/DS/HA preparation.
Heparinase I, II, III digestion of samples for HS analysis
Note
Perform this section only for samples that require HS disaccharide analysis.
Note: NEB enzymes are supplied with a 10 x Heparinase Buffer that have salts that are not compatible with downstream HILIC-MS/MS analysis. Therefore a different Heparinase Buffer is prepared and used here.
Add 5 µL 10 x Heparinase Buffer to GAG enriched samples
for buffer preparation
for samples
Thaw the amount of each heparinase enzyme (Hep I, Hep II, Hep III) required for all samples
Bacteroides Heparinase I - 600 unitsNew England BiolabsCatalog #P0735L Bacteroides Heparinase II - 200 unitsNew England BiolabsCatalog #P0736L Bacteroides Heparinase III - 35 unitsNew England BiolabsCatalog #P0737L
Note
Each heparinase enzyme is stored at -80oC neat in 2-20 μL aliquots, only thaw the volume you need.
Each sample requires 1 μL of each enzyme.
Therefore for 12 samples, thaw 12 μL of each enzyme.
Combine the Hep I, Hep II and Hep III into one tube = Heparinase Enzyme Mix.
Spin briefly to collect at the bottom of the tube if needed.
Note
I.e. if there are 12 samples then combine 12 μL Hep I, 12 μL Hep II and 12 μL Hep III.
Add 3 µL Heparinase Enzyme Mix to each sample
Mix by pipetting or vortexing. Spin briefly to collect at the bottom of the tube
Incubate at 30 °C overnight, with mixing at 2000 rpm
Chondroitinase ABC digestion for CS/DS/HA analysis
Note
Perform this section only for samples that require CS/DS/HA disaccharide analysis.
Add 5 µL 10 x Chondrotinase Buffer to GAG enriched samples
for buffer preparation
for samples
Thaw the amount of Chondrotinase ABC (ChABC) enzyme required for all samples
Chondroitinase ABC from Proteus vulgarisMerck MilliporeSigma (Sigma-Aldrich)Catalog #C2905-10UN
Note
ChABC is stored at -80oC in 10 μL aliquots at 10 mU/μL in 50 mM sodium acetate pH 7.
Only thaw the volume you need.
Each sample requires 1 μL of ChABC at 10 mU/μL (before dilution).
Therefore for 12 samples, thaw 12 μL of 10 mU/μL ChABC.
If there are multiple aliquots, combine them into one tube
Dilute ChABC to 2.5 mU/μL by adding 3 parts ddH2O
Note
This step allows a higher volume of enzyme to be pipetted into each sample, reducing pipetting error.
For 12 samples, prepare 2.5 mU/μL as follows:
- Take 12 µL 10 mU/μL ChABC
- Add 36 µL ddH2O
- Mix well
Add 4 µL 2.5 mU/μL ChABC to each sample
- (Therefore the amount added is 10 mU/4 μL)
Mix by pipetting or vortexing. Spin briefly to collect at the bottom of the tube.
Incubate at 37 °C overnight with mixing at 2000 rpm
GAG disaccharide clean-up
Prepare 1 μg/mL Internal Standard
Note
This section, and all subsequent sections, apply to both HS and CS/DS/HA disaccharide preparations.
The Internal Standard is spiked into samples (and standards in a separate protocol) and serves to to correct for variability in the extraction and analysis from this point.
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The internal standard stock is stored at -20 oC in 10 µL aliquots of 1 mg/mL in ddH2O.
Thaw the 1 mg/mL Internal Standard Stock∆UA-2S GlcNCOEt-6S (I-P) internal standard disaccharideIduronCatalog #HD009
Dilute the Internal Standard Stock to make a 10 μg/mL Internal Standard Solution (1 in 100 dilution):
- To the 10 µL aliquot add 990 µL ddH2O and mix well
Dilute the 10 μg/mL Internal Standard Solution to make 1 μg/mL Internal Standard Solution (1 in 10 dilution):
- To a fresh microcentrifuge tube add 100 µL 10 μg/mL Internal Standard Solution
- Add 900 µL ddH2O
- Mix well
- Store at4 °C when not in use
Centrifuge all samples after GAG digestion at 15000 rpm for 15 minutes at 4 °C
Solid Phase Extraction (SPE) clean-up and Internal Standard spike
Note
This steps acts as a scrub - disaccharides are hydrophilic and will not bind to the stationary phase of the HLB plate, whilst hydrophobic proteins and contaminants will bind and be removed.
Equipment
Oasis HLB 96-well µElution Plate, 2 mg Sorbent per Well, 30 µm, 1/pk
NAME
Sample preparation and filtration
TYPE
Waters
BRAND
186001828BA
SKU
Over a waste collection plate, pre-wash the number of wells needed (one well per sample) with 100 µL ddH2O and centrifuge to waste at 1000 rpm for 2 minutes, or until wells are dry
- Discard the wash
Transfer HLB plate on top of a clean collection plate
Assign a well to each sample
| A | B | C | D | E | F | G | H | I | J | K | |
Load each sample supernatant into a HLB plate well. for samples
- Keep the lid of the microcentrifuge tubes for freeze-drying later. Carefully pierce several holes in the lid and remove the lid from the tube.
Carefully use a multichannel pipette to add 8 µL 1 μg/mL Internal Standard Solution to each well (ensure pipetting accuracy across all channels).
for Internal Standard Solution preparation
Note
Here 8 ng internal standard is added. At the end of the sample preparation the sample will be in 20 μL water, and so samples will have 8 ng/20 μL internal standard (400 ng/mL)
Mix each well by pipetting up/down several times
Centrifuge at 100-500 x g for 3 minutes (aim for the sample to come through slowly to boost interaction with the solid phase of the well)
- Check the wells to see if the volume has come through
- If there is still volume repeat further spins at 500-2000 x g rpm for 3-5 minutes
- Or until all volume has come through the wells and into the collection plate
Add 50 µL ddH2O to wells and centrifuge at 100-2000 x g rpm to collect with the sample flow through. Centrifuge until the wells are dry.
For each sample transfer the collected flow through to labelled 2 mL safelock tube.
- Add the lids with pierced holes in from earlier for lids
- (The pierced lids enable lyophilisation)
- Leave the original lid still attached to the tube for later
Freeze the samples at -80 °C or on dry ice briefly
Freeze-dry the samples. Then cap them with the unpierced lid of the microtube.