Apr 28, 2026
Disruption of biological tissues, and amounts required, for GAG disaccharide preparation
- 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. Disruption of biological tissues, and amounts required, for GAG disaccharide preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl88pwrl2w/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: August 19, 2025
Last Modified: April 28, 2026
Protocol Integer ID: 224952
Keywords: Sample disruption, GAG disaccharide analysis, gag disaccharide preparation sample preparation, gag disaccharide analysis, free gag disaccharides from biological tissue, gag disaccharide preparation sample preparation of label, gag disaccharide preparation this protocol, gag disaccharide preparation, gag disaccharide data, subsequent normalisation of gag disaccharide data, gag disaccharide standard, gag disaccharide, free gag disaccharide hilic, free gag disaccharide, disaccharide, biological tissues equimolar stock solution preparation, ms analysis from biological tissue, sample preparation, sample preparation of label, samples for analysis hilic, diverse types of biological material, preparation, ms data acquisition, disruption of biological tissue, protein amount
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 for the disruption of diverse types of biological material, prior to GAG disaccharide preparation. Guidelines are provided for the amount of starting material required. Suggestions are also made for measurements that may facilitate subsequent normalisation of GAG disaccharide data (e.g. to protein amount).
This is the first protocol in a collection, which documents the details of all steps required for label-free GAG disaccharide HILIC-MS/MS analysis from biological tissues:
- Disruption of biological tissues for GAG disaccharide preparation
The disrupted material generated here can then be prepared for GAG disaccharide analysis, vialled and analysed according to protocols 3-5 in the collection.
This information provided here is not an exhaustive list and the protocol will evolve over time as more types of material and methods of disruption are studied.
Guidelines
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.
In addition, for human samples ensure the work is covered by a licence from the HTA.
Cells
Note
Number of cells required for GAG analysis by HILIC-MS/MS*
*Based on observations with CHO cell data. However, this will vary with cell type.
For comprehensive characterisation of GAG disaccharides (in order to detect as many of the disaccharides as possible), the recommended number of cells is:
- 1 million cells
For identification of the most abundant disaccharides (in order to compare relative GAG disaccharide levels across samples), the recommended number of cells is:
- > 50000 cells for CS/DS/HA‡
- > 50000 cells for HS‡
‡ The numbers given here are initial guidelines based on observations during method development. Lower numbers of cells are likely to provide data, but the the lower limit of cells required has not been fully determined, and will depend on cell type.
Disruption of cells
Here, cells are counted and prepared as pellets or lysates for subsequent use in the GAG disaccharide sample preparation protocol. Optimisation of homogenisation may be required for different cell types, and alternative homogenisation techniques may be possible but have not been tested at the time of publication of this protocol.
Safety
Samples should be handled in biological safety cabinets to protect the user from any risk of infectious agents.
Count the cells
Collect the required number of cells. Current requirements are given above
- Note the number of cells taken forward (this is recommended for subsequent normalisation of GAG disaccharide data to number of cells, to enable relative quantification across samples)
Pellet the cells by centrifugation, washing twice in PBS
For example:
- Recover cells by centrifugation at 300 x g for 3 minutes
- Discard the media
- Gently resuspend in PBS
- Recover cells by centrifugation at 300 x g for 3 minutes
- Discard the PBS
- Gently resuspend in PBS
- Recover cells by centrifugation at 300 x g for 3 minutes
On the final wash, remove the majority of the liquid
This step has not yet been optimised but guidelines are given here:
- Freeze the cell pellet (for example at -20 oC) and submit for GAG disaccharide sample preparation.
or
- Generate cell lysate by sonication (for example in a sonicator bath for ~ 20 minutes) in a small volume of 50 mM ammonium acetate pH 7.5. Cell lysates could then be frozen at -20 oC and submitted for GAG disaccharide sample preparation.
Frozen tissue (100 - 300 mg)
Note
Amount of tissue required for GAG disaccharide analysis by HILIC-MS/MS
For comprehensive characterisation of GAG disaccharides (in order to detect as many of the disaccharides as possible), the recommended amount of tissue is:
- ≥ 5 mg tissue
For identification of the most abundant disaccharides (in order to compare relative GAG disaccharide levels across samples), the recommended amount of tissue is:
- ≥ 0.5 mg tissue*
* The amounts of tissue given here are initial guidelines based on observations during method development. Lower amounts of tissue are likely to provide data, but the the lower limit of tissue required has not been fully determined, and will depend on tissue type.
Disruption of tissue 100- 300 mg
Where biological tissues are 100 - 300 mg is it recommended to follow sample disruption as described in this section. If samples are > 300 mg then dissection should be performed to generate samples of 100 - 300 mg. For samples < 100mg see other sections in this protocol.
Alternative disruption techniques may be possible but have not been tested at the time of publication of this protocol.
Safety
Tissue should be handled in a class II microbiological safety cabinet to protect the user from any risk of infectious agents.
Weigh tissue and record the weight
- If tissue is 100-300 mg record the weight (for subsequent normalisation of GAG disaccharide data for the amount of tissue, facilitating comparison of relative GAG amounts across tissues)
- If tissue is > 300 mg then dissect the tissue to obtain 100-300 mg pieces (and record the weights)
Disrupt the tissue using the Covaris CP02 CryoPREP Automated Dry Pulveriser
Use the appropriate consumables and adapters
- The following are suitable for 30 - 300 mg tissue:
Equipment
tissueTUBE TT1 Extra Thick
NAME
Covaris
BRAND
520007
SKU
LINK
Equipment
tissueTUBE TT1 - milliTUBE Adapter
NAME
Covaris
BRAND
520142
SKU
LINK
Equipment
tissueTUBE TT1 Insertion Tool
NAME
Covaris
BRAND
500159
SKU
LINK
Place the weighed tissue inside the tissueTUBE
Assemble the tissueTUBE onto the milliTUBE using the milliTUBE adapter
Unscrew the milliTUBE by a quarter turn
Place the milliTUBE into the Insertion Tool
Assembly of consumables for 100-300 mg tissue pulverisation by CryoPREP
Hold the assembly by the Insertion Tool and submerge the tissueTUBE into liquid nitrogen
Place the assembly on the cryoPREP and close the lid
- Choose an appropriate impact level setting for the type of tissue
- Asset URL:
Press the Activate button to deliver impact to the sample
- Consider repeating this step twice if pulverisation is not sufficient
Recover the disrupted tissue from the tissueTUBE into the milliTUBE
For easy transfer, perform this as follows:
- Following cryoPREP, the powdered tissue will be clumped into a pellet within the tissueTUBE
- Hold the assembly by the Insertion Tool and dip the tissueTUBE containing the powdered tissue into liquid nitrogen briefly
- On a lab bench, use an object such as a 50 mL Duran Bottle to roll over the tissueTUBE, disrupting the clumped tissue into a powder that can be easily transferred into milliTUBE by inverting the tissueTUBE over the milliTUBE and flicking the tissueTUBE to transfer to contents to the milliTUBE.
Either:
- Submit samples for GAG disaccharide preparation
- Store samples for later processing for GAG disaccharide preparation. Storage conditions may require optimisation for each sample type. Unoptimised guidelines are to freeze at -80 oC.
Frozen tissue (~5 - 100 mg)
Note
Amount of tissue required for GAG disaccharide analysis by HILIC-MS/MS
For comprehensive characterisation of GAG disaccharides (in order to detect as many of the disaccharides as possible), the recommended amount of tissue is:
- ≥ 5 mg tissue
For identification of the most abundant disaccharides (in order to compare relative GAG disaccharide levels across samples), the recommended amount of tissue is:
- > 0.5 mg tissue*
* The amounts of tissue given here are initial guidelines based on observations during method development. Lower amounts of tissue are likely to provide data, but the the lower limit of tissue required has not been fully determined, and will depend on tissue type.
Disruption of tissue 5- 100 mg
It is at the users discretion whether to assess the powdered tissue, or generate a homogenate by extracting the material in a solution, such as PBS.
For generating powdered tissue from 5 - 100 mg starting tissue, the Covaris cryoPREP Automated Dry Pulverisation is described in this section.
For generating tissue homogenates from 5 - 100 mg starting tissue, bead homogenisation by a Qiagen TissueLyser II is detailed in this section.
Alternative disruption techniques may be possible but have not been tested at the time of publication of this protocol.
For samples > 100 mg see preceding sections in this protocol; for < 5 mg see subsequent sections in this protocol.
Safety
Tissue should be handled in a class II microbiological safety cabinet to protect the user from any risk of infectious agents.
Weigh tissue and record the weight
- This is for subsequent normalisation of GAG disaccharide data for amount of tissue, to facilitate relative quantification of GAGs across tissue samples.
Option for generating powdered tissue - Disrupt the tissue using the Covaris CP02 CryoPREP Automated Dry Pulveriser
Use the appropriate consumables and adapters
- The following are suitable for 5 - 100 mg tissue:
Place the weighed tissue inside the tissueTUBE
Assemble the tissueTUBE onto the milliTUBE
Unscrew the milliTUBE by a quarter turn
Place the milliTUBE into the Insertion Tool
Assembly of consumables for 5-100 mg tissue pulverisation by CryoPREP
Hold the assembly by the Insertion Tool and submerge the tissueTUBE into liquid nitrogen
Place the assembly on the cryoPREP and close the lid
- Choose an appropriate impact level setting for the type of tissue
- Asset URL:
Press the Activate button to deliver impact to the sample
- This step can be repeated if pulverisation is not sufficient.
Recover the disrupted tissue from the tissueTUBE into the milliTUBE
For easy transfer, perform this as follows:
- Following cryoPREP, the powdered tissue will be clumped into a pellet within the tissueTUBE
- Hold the assembly by the Insertion Tool and dip the tissueTUBE containing the powdered tissue into liquid nitrogen briefly
- On a lab bench, use an object such as a 50 mL Duran Bottle to roll over the tissueTUBE, disrupting the clumped tissue into a powder that can be easily transferred into milliTUBE by inverting the tissueTUBE over the milliTUBE and flicking the tissueTUBE to transfer to contents to the milliTUBE.
Either:
- Submit samples for GAG disaccharide preparation
- Store samples for later processing for GAG disaccharide preparation. Storage conditions may require optimisation for each sample type. Unoptimised guidelines are to freeze at -80 oC.
Option for generating tissue homogenates - Bead homogenisation via Qiagen TissueLyser II
- Detailed protocols are provided in the supplier user manual. Guidelines are provided in this section.
Place the weighed tissue inside a 2 mL microfuge tube
Add a 5 mm stainless steel ball
- The stainless steel balls are reusable, stored in 70% and should be washed in water before use
Equipment
Stainless Steel 5 mm beads
NAME
Qiagen
BRAND
69989
SKU
Add PBS to the tube
- Use between 100 - 400 μL PBS per tube
- The volume of PBS can be optimised for each tissue type
Place tubes in the TissueLyser Adapter for 24 x 2 mL tubes
- Balance the tubes across both adapters
- Add the adapter lids and clamp onto the TissueLyser II
Homogenise at a frequency of 30.s for 4 x 30 seconds
- This step can be optimised for each tissue type
Transfer the homogenised material to a fresh tube, leaving behind the stainless steel ball
- Wash the stainless steel ball in 70% ethanol and water
- Store in 70% ethanol for reuse
Generate either (a) soluble homogenate or (b) both insoluble and soluble material for analysis. All samples to be compared should be treated consistently.
Option (a) soluble homogenate:
- Centrifuge the homogenate (e.g. at 10000 rpm for 10 minutes) and recover the supernatant to collect only the soluble homogenate
Option (b) for both insoluble and soluble material:
- Provide all material without centrifugation
Either:
- Submit samples for GAG disaccharide preparation
- Store samples for later processing for GAG disaccharide preparation. Storage conditions may require optimisation for each sample type. Unoptimised guidelines are to freeze at -80 oC.
Frozen tissue biopsies (<5 mg)
Note
Amount of tissue required for GAG disaccharide analysis by HILIC-MS/MS
For comprehensive characterisation of GAG disaccharides (in order to detect as many of the disaccharides as possible), the recommended amount of tissue is:
- ≥ 5 mg tissue
For identification of the most abundant disaccharides (in order to compare relative GAG disaccharide levels across samples), the recommended amount of tissue is:
- ≥ 0.5 mg tissue*
* The amounts of tissue given here are initial guidelines based on observations during method development. Lower amounts of tissue are likely to provide data, but the the lower limit of tissue required has not been fully determined, and will depend on tissue type.
Disruption of tissue < 5 mg
This section covers tissue that is ~ 5 mg or less.
Here, homogenisation of the tissue is performed by bead homogenisation with a Qiagen TissueLyser II. Other forms of homogenisation may be possible but have not been tested at the time of publication of this protocol.
For samples > 5 mg see preceding sections in this protocol.
Safety
Tissue should be handled in a class II microbiological safety cabinet to protect the user from any risk of infectious agents.
Weigh tissue and record the weight
- This is for subsequent normalisation of GAG disaccharide data for amount of tissue, to facilitate relative quantification of GAGs across tissue samples.
Bead homogenisation via Qiagen TissueLyser II
- Detailed protocols are provided in the supplier user manual. Guidelines are provided in this section.
Place the weighed tissue inside a 2 mL microfuge tube
Add a 5 mm stainless steel ball
- The stainless steel balls are reusable, stored in 70% and should be washed in water before use
Equipment
Stainless Steel 5 mm beads
NAME
Qiagen
BRAND
69989
SKU
Add PBS to the tube
- Use between 100 - 200 μL PBS per tube
- The volume of PBS can be optimised for each tissue type
Place tubes in the TissueLyser Adapter for 24 x 2 mL tubes
- Balance the tubes across both adapters
- Add the adapter lids and clamp to the TissueLyser II
Homogenise at a frequency of 30.s for 4 x 30 seconds
- This step can be optimised for each tissue type
Transfer the homogenised material to a fresh tube, leaving behind the stainless steel ball
- Wash the stainless steel ball in 70% ethanol and water
- Store in 70% ethanol for reuse
Generate either (a) soluble homogenate (after centrifugation) or (b) both insoluble and soluble material for analysis. All samples to be compared should be treated consistently.
Option (a) soluble homogenate:
- Centrifuge the homogenate (e.g. at 10000 rpm for 10 minutes) and recover the supernatant to collect only the soluble homogenate
Option (b) for both insoluble and soluble material:
- Provide all material without centrifugation
Either:
- Submit samples for GAG disaccharide preparation
- Store samples for later processing for GAG disaccharide preparation. Storage conditions may require optimisation for each sample type. Unoptimised guidelines are to freeze at -80 oC.
FFPE scrolls
Note
Amount of FFPE tissue required for GAG disaccharide analysis by HILIC-MS/MS
The amount of tissue per FFPE block varies greatly with sample type (e.g. population differences, organ type (e.g. liver vs brain) and species (e.g. mouse vs human)).
At the time of publication of this protocol, comprehensive characterisation of GAG disaccharides has been demonstrated from the following amounts of FFPE material:
- 3 x 25
μm FFPE scrolls of mouse kidney per sample - 1 x 10 μm FFPE scroll of human kidney biopsy
Disruption of tissue FFPE scrolls
Here, tissue is liberated from FFPE scrolls by bead homogenisation with a Qiagen TissueLyser II, decrosslinked by incubation at 98 oC and homogenised by further bead homogenisation.
Alternative techniques for the liberation of tissue homogenates from FFPE scrolls techniques may be possible but have not been tested at the time of publication of this protocol.
Normalisation for relative quantification of GAGs per FFPE sample
At the time of publication of this protocol, normalisation for FFPE scrolls is under optimisation.
Options include:
- Estimation of tissue area per sample by calculation of tissue cross-sectional areas from FFPE blocks (FFPE scrolls thickness should be fixed for all samples to be compared);
- Calculation of protein amount in tissue after liberation and homogenisation from FFPE sections using Bicinchoninic Acid (BCA) Assay. Note: the amount of tissue required for the BCA assay may be greater than the amount required for GAG analysis by HILIC MS/MS, therefore a greater number of FFPE scrolls might be required if this normalisation approach is selected.
Safety
During homogenisation steps, avoid opening tubes outside a class II microbiological safety cabinet to protect the user from any risk of aerosolised infectious agents.
Section FFPE blocks into scrolls/curls
Ideally remove excess wax from tissue block before sectioning
Optional: for normalisation to tissue area take a photo of FFPE block next to a ruler
- Use this to estimate the tissue area per sample
- Tissue area could then be used to normalise relative amounts of GAG disaccharide by tissue area, to facilitate the comparison of relative amount of GAGs across FFPE samples
Use a microtome to section FFPE block into scrolls/curls and collect the required number into a 2 mL microfuge tube
- The thickness of sections can be optimised per sample type
- The number of sections can be optimised per sample type
- As a guideline 1 x 25 μm scroll and 3 x 10 μm scrolls may be enough for GAG disaccharide analysis, but more material may be required for protein measurement by BCA analysis as well (which is another means of normalisation)
Bead homogenisation via Qiagen TissueLyser II
- Detailed protocols are provided in the supplier user manual. Guidelines are provided in this section
Add a 5 mm stainless steel ball to the 2 mL microcentrifuge tube
- The stainless steel balls are reusable, stored in 70% and should be washed in water before use
Equipment
Stainless Steel 5 mm beads
NAME
Qiagen
BRAND
69989
SKU
Add 200 μL PBS to the tube
Place tubes in the TissueLyser Adapter for 24 x 2 mL tubes
- Balance the tubes across both adapters
- Add the adapter lids and clamp to the TissueLyser II
Homogenise at a frequency of 30.s for 4 x 30 seconds
- This step can be optimised for each tissue type
Decrosslink and melt wax to aid further tissue disruption
- Heat samples to 98 oC for 30 minutes
- Half way through invert tubes/vortex briefly to mix (careful not to allow lids to open)
Repeat homogenisation and decrosslinking steps:
- Homogenise at a frequency of 30.s for 2 x 30 seconds
- Heat samples to 98 oC for 30 minutes
- Half way through invert tubes/vortex briefly to mix (careful not to allow lids to open)
Centrifuge at 300 x g for 1 minute
- During centrifugation the wax should pellet/stick to walls of microcentrifuge tube, whilst the homogenised tissue will remain in the PBS
- Gently pipette the PBS containing the tissue homogenate up and down to ensure recovery of any sedimented tissue from around the stainless steel bead
- Withdraw the tissue homogenate and place inside a fresh microcentrifuge tube
Optional: If there is still tissue in the tube or near the bead then further processing could be performed:
- Add another 200 μL PBS and repeat the homogenisation (30.s for 2 x 30 seconds), decrosslinking (98 oC for 30 minutes) and centrifugation (300 x g for 1 minute) steps in the original 2 mL microcentrifuge tube
- Combine this homogenate with the first to make one sample of 400 μL
- Ensure the same steps are performed for all samples in a batch
Consider performing protein concentration measurement (eg BCA assay)
- This is for subsequent normalisation of GAG disaccharide data to the amount of protein in sample, to facilitate relative quantification of GAGs across tissue samples.
Either:
- Submit samples for GAG disaccharide preparation
- Store samples for later processing for GAG disaccharide preparation. Storage conditions may require optimisation for each sample type. Unoptimised guidelines are to freeze at -80 oC.
Wash stainless steel beads in water and store in 70% ethanol.
Acknowledgements
Thank you to Rebecca Dodd and Judi Allen (Manchester Cell-Matrix Centre, University of Manchester, UK) for the use of the Qiagen TissueLyser II.