Apr 28, 2026

Equimolar Stock Solution of GAG disaccharide standards

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld6knxg5b/v1
  • 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
H Davies-Strickleton
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ld6knxg5b/v1
Protocol Citation: H Davies-Strickleton, James Allsey, Douglas Dyer, David Knight 2026. Equimolar Stock Solution of GAG disaccharide standards. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld6knxg5b/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: November 27, 2025
Last Modified: April 28, 2026
Protocol  Integer ID: 233659
Keywords: GAG disaccharides, Heparan sulphate, Chondroitin sulphate, Dermatan sulphate, Hyaluronan, GAG characterisation, GAG disaccharide composition, GAG quantification, HILIC-MS/MS, gag disaccharide preparation sample preparation, gag disaccharide preparation sample preparation of label, equimolar stock solution of gag disaccharide standard, gag disaccharide preparation, gag disaccharide standard, free gag disaccharides from biological tissue, relative quantification of gag disaccharide, free gag disaccharide hilic, gag disaccharide, free gag disaccharide, mixture of disaccharide, biological tissues equimolar stock solution preparation, disaccharide, ms analysis from biological tissue, preparation of an equimolar stock solution, sample preparation of label, sample preparation, samples for analysis hilic, ms data acquisition, uv absorbance, preparation, equimolar stock solution, type of gag
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 describes the preparation of an Equimolar Stock Solution of GAG disaccharide standards. For each type of GAG (HS or CS/DS/HA) a mixture of disaccharides at equal molar concentration, as defined by their UV absorbance at 232 nm, is prepared.

This is the third protocol in a collection, which documents the details of all steps required for label-free GAG disaccharide HILIC-MS/MS analysis from biological tissues:
  1. Disruption of biological tissues for GAG disaccharide preparation
  2. Sample preparation of label-free GAG disaccharides from biological tissues
  3. Equimolar Stock Solution preparation of GAG disaccharide standards
  4. Vialling of Standards and Samples for analysis
  5. HILIC-MS/MS data acquisition and processing

The Equimolar Stock Solution prepared here is subsequently vialled and analysed by HILIC-MS/MS (protocols 4-5). This enables relative quantification of GAG disaccharides from biological tissues (that are prepared in protocols 1-2).
Protocol materials
∆UA – GalNAc, 6SIduronCatalog #CD003
UA-GlcNAcIduronCatalog #HD006
UA,2S-GlcNS,6SIduronCatalog #HD001
∆UA – GalNAc, 4SIduronCatalog #CD002
∆UA, 2S – GalNAc, 4SIduronCatalog #CD005
∆UA, 2S – GalNAc, 6S IduronCatalog #CD006
UA-GlcNS,6SIduronCatalog #HD004
UA-GlcNSIduronCatalog #HD005
UA,2S-GlcNAcIduronCatalog #HD007
∆UA – GalNAc, 4S, 6SIduronCatalog #CD004
∆UA(1-3) – GlcNAcIduronCatalog #HA02
UA,2S-GlcNSIduronCatalog #HD002
UA,2S-GlcNAc,6SIduronCatalog #HD003
UA-GlcNAc,6SIduronCatalog #HD008
∆UA – GalNAcIduronCatalog #CD001
∆UA, 2S – GalNAcIduronCatalog #CD008
Disaccharide Stock preparation

Note
Steps 1-9 of this protocol refer to the preparation of freshly purchased disaccharide standards into Disaccharide Stocks

  • This involves reconstitution of purchased lyophilised standards, their measurement by UV absorption at 232 nm, and determination of correction factors required for generating a mixture of disaccharide standards at equal molar concentration, as defined by their UV absorption at 232 nm.

  • Once prepared, the disaccharide can be stored at -20oC.

Steps 10 onwards describe the dilution steps required to generate the Equimolar Stock Solution (a mixture of disaccharides at equal molar concentration, defined by their UV absorption at 232 nm)


Disaccharide standards are purchased as lyophilised material of either 0.5 mg or 1 mg.

HS disaccharide standards:
  • UA,2S-GlcNS,6SIduronCatalog #HD001 (1 mg)
  • UA,2S-GlcNSIduronCatalog #HD002 (1 mg)
  • UA,2S-GlcNAc,6SIduronCatalog #HD003 (1 mg)
  • UA-GlcNS,6SIduronCatalog #HD004 (1 mg)
  • UA-GlcNSIduronCatalog #HD005 (1 mg)
  • UA-GlcNAcIduronCatalog #HD006 (1 mg)
  • UA,2S-GlcNAcIduronCatalog #HD007 (1 mg)
  • UA-GlcNAc,6SIduronCatalog #HD008 (1 mg)

CS disaccharide standards:
  • ∆UA – GalNAcIduronCatalog #CD001 (1 mg)
  • ∆UA – GalNAc, 4SIduronCatalog #CD002 (1 mg)
  • ∆UA – GalNAc, 6SIduronCatalog #CD003 (1 mg)
  • ∆UA – GalNAc, 4S, 6SIduronCatalog #CD004 (0.5 mg)
  • ∆UA, 2S – GalNAc, 4SIduronCatalog #CD005 (0.5 mg)
  • ∆UA, 2S – GalNAc, 6S IduronCatalog #CD006 (0.5 mg)
  • ∆UA, 2S – GalNAcIduronCatalog #CD008 (0.5 mg)
  • ∆UA(1-3) – GlcNAcIduronCatalog #HA02 (1 mg)


Add 1 mL ddH2O to each vial to give Disaccharide Stocks of provisional* concentrations of 0.5 - 1 mg/mL.

*Note: these stocks are considered as provisional concentrations (i.e. prior to correction by UV absorption at 232 nm), and are referred to as such below.

Mix well by vortexing briefly

Aliquot and store at -20 oC until use

  • Avoid repeat freeze-thaw cycles

Measure UV absorption at 232 nm of 100 μM Disaccharide Solutions

Note
Rationale for measurement of UV absorption of disaccharides

Measurement of freshly prepared Disaccharide Stocks by UV absorption at 232 nm provides the means to normalise Disaccharide Stock concentrations, and ultimately generate mixtures of disaccharide standards at equal molar concentration (Equimolar Stock Solution).

  • This was found to be necessary based on the observation that multiple lyophilised vials of the same disaccharide standard, after reconstitution, were found to contain differing concentrations of material (based on UV and MS responses).



Here, each Disaccharide Stock is prepared to a provisional molar concentration and analysed by UV absorbance at 232 nm.

  • Correction factors are then calculated by dividing the UV absorbance of each standard by the UV absorbance obtained from the HD002 (HS) or CD001 (CS/DS/HA). HD002 and CD001 were selected as the reference standards due to their consistency in an initial multi-batch comparison.

  • The correction factors are used to determine the UV-corrected molar concentration of the Disaccharide Stocks.

  • In steps 10 onwards the Disaccharide Stocks are diluted (based on their UV-corrected molar concentrations) to generate the Equimolar Stock Solution (a mixture of disaccharides at equal molar concentration, defined by their UV absorption at 232 nm)


Convert Disaccharide Stock concentration (provisional mg/mL) to provisional molarity (mM)


Note
Note: provisional refers to the concentrations prior to UV correction



Conversion to mM

The formula below was used to convert mg/mL provisional concentrations to mM provisional concentrations:


where:
M = Molarity; mol/L
C = Concentration: 1 mg/mL = 1 g/L
MW = Molecular weight; g/mol

For example for HD001:




HS Disaccharide Stocks (provisional mM concentration):
ABCDE
Analyte nameMWProvisional amount in vial (mg)Provisional concentration of Disaccharide Stock (C, mg/mL)Provisional concentration of Disaccharide Stock (mM; mmol/L)
HD001665111.504
HD002563111.776
HD003605111.653
HD004563111.776
HD005461112.169
HD006401112.494
HD007503111.988
HD008503111.988


CS/DS/HA Disaccharide Stocks (provisional mM concentration):
ABCDE
Analyte nameMWProvisional amount in vial (mg)Provisional concentration of Disaccharide Stock (C, mg/mL)Provisional concentration of Disaccharide Stock (mM; mmol/L)
CD001401112.494
CD002503111.988
CD003503111.988
CD0046050.50.50.826
CD0056050.50.50.826
CD0066050.50.50.826
CD0085030.50.50.994
HA02401112.494



Preparation of HS Disaccharide Solutions of 100 μM (provisional concentration)

For each HS disaccharide, use the table below to generate 200 μL of 100 μM (provisional concentration).
  • See note for calculations

For each HS disaccharide:
  • Label a microfuge tube
  • Add the volume of ddH2O shown in column E
  • Add the volume of 1 mg/mL (provisional concentration) Disaccharide Stock shown in column D
  • Mix well
  • Prepare in triplicate


ABCDE
Analyte nameProvisional concentration of Disaccharide Stock (mg/mL)Provisional concentration of Disaccharide Stock (mM; mmol/L)Volume of Disaccharide Stock required (V1, uL)Volume of ddH2O required (uL)
HD00111.50413.3186.7
HD00211.77611.3188.7
HD00311.65312.1187.9
HD00411.77611.3188.7
HD00512.1699.2190.8
HD00612.4948.0192.0
HD00711.98810.1189.9
HD00811.98810.1189.9


Note
Volumes can be adjusted according to the following approach:

1. The following formula was used to calculate the volume of each Disaccharide Stock required to prepare provisional 100 μM (0.1 mM) disaccharide solutions at a volume of 200 μL.


rearranged provides:

where:
C1 = Starting concentration (mM) for mM concentrations of stocks
V1 = Volume of stock needed ( μL)
C2 = Desired concentration (mM)
V2 = Desired total volume (μL)



For example for HD001:





2. Calculate the volume of water required to add to make the volume up to 200 μL (= 200 - volume of Disaccharide Stock)


3. Repeat steps 1 and 2 for each disaccharide


Preparation of CS/DS/HA Disaccharide Solutions of 100 μM (provisional concentration)

For each CS/DS/HA disaccharide, use the table below to generate 200 μL of 100 μM (provisional concentration).
  • See note for calculations.

For each CS/DS/HA disaccharide:
  • Label a microfuge tube
  • Add the volume of ddH2O shown in column E
  • Add the volume of 0.5-1 mg/mL (provisional concentration) Disaccharide Stock shown in column D
  • Mix well
  • Prepare in triplicate


ABCDE
Analyte nameProvisional concentration of Disaccharide Stock (mg/mL)Provisional concentration of Disaccharide Stock (mM; mmol/L)Volume of Disaccharide Stock required (V1, uL)Volume of ddH2O required (uL)
CD00112.4948.0192.0
CD00211.98810.1189.9
CD00311.98810.1189.9
CD0040.50.82624.2175.8
CD0050.50.82624.2175.8
CD0060.50.82624.2175.8
CD0080.50.99420.1179.9
HA0212.4948.0192.0


Note
Volumes can be adjusted according to the following approach:

1. The following formula was used to calculate the volume of each Disaccharide Stock required to prepare provisional 100 μM (0.1 mM) disaccharide solutions at a volume of 200 μL.


rearranged provides:

where:
C1 = Starting concentration (mM) for mM concentrations of stocks
V1 = Volume of stock needed ( μL)
C2 = Desired concentration (mM)
V2 = Desired total volume (μL)



For example for CD001:





2. Calculate the volume of water required to add to make the volume up to 200 μL (= 200 - volume of Disaccharide Stock)


3. Repeat steps 1 and 2 for each disaccharide

Measure the provisional 100 μM Disaccharides Solutions for UV absorption at 232 nm

Here, a Molecular Devices SpectraMax i3x Multi-Mode Plate Reader (Softmax Pro 7.1.2) was used to measure UV absorption at 232 nM:
  • Add 200 μL of each 100 μM Disaccharide Solution to individual wells of a UV-transparent 96 well plate, in triplicate (within the software assign the wells as unknowns)
  • To a separate well, add 200 μL ddH2O as a blank (within the software assign the wells as blanks)
  • Measure the UV absorption at 232 nM
  • For each disaccharide, average the UV absorbance values across the triplicate measurements
  • Recover the Disaccharide Solutions and freeze at -20oC for future use

Equipment
UV-STAR® MICROPLATE, 96 WELL, COC, F-BOTTOM
NAME
96 well plate
TYPE
Greiner
BRAND
655801
SKU
https://shop.gbo.com/en/england/products/bioscience/microplates/uv-star-microplates/96-well-uv-star-microplate/655801.html
LINK



For each GAG type (HS or CS/DS/HA) calculate UV correction factors and UV-corrected stock concentrations

  • Choose a disaccharide to use as a reference disaccharide. For example, for the currently working disaccharide standards in our lab HD002 and CD001 were selected due to their consistency in an initial multi-batch comparison.
  • Divide the absorbance of each disaccharide by the absorbance obtained from the reference disaccharide (HD002 for HS disaccharides and CD001 for CS/DS/HA disaccharides) = correction factor
  • Multiply the provisional molar Disaccharide Stock concentrations by the correction factor to obtain a UV-corrected molar Disaccharide Stock concentration

For example, these correction factors and UV-corrected concentrations apply to our current working HS Disaccharide Stocks:

ABCD
Analyte nameProvisional concentration of Disaccharide Stock (mM)Correction factor determined by UV absorbance at 232 nmUV-corrected concentration of Disaccharide Stock (mM)
HD0011.5042.6443.976
HD0021.7761.0001.776
HD0031.6531.0191.685
HD0041.7761.1211.991
HD0052.1690.9021.958
HD0062.4941.6124.021
HD0071.9880.8981.785
HD0081.9880.9041.797

For example, these correction factors and UV-corrected concentrations apply to our current working CS/DS/HA Disaccharide Stocks:

ABCD
Analyte nameProvisional concentration of Disaccharide Stock (mM)Correction factor determined by UV absorbance at 232 nmUV-corrected concentration of Disaccharide Stock (mM)
CD0012.4941.0002.494
CD0021.9881.4232.829
CD0031.9881.0782.143
CD0040.8260.7970.658
CD0050.8260.6890.569
CD0060.8260.9860.815
CD0080.9940.6940.690
HA022.4940.5811.448



The UV-corrected concentrations can then be used to prepare equimolar mixtures of disaccharides as detailed below.
Preparation of Equimolar Stock Solution

Note
Here, for each GAG type (HS or CS/DS/HA), each disaccharide is combined to provide an Equimolar Stock Solution (a mixture of disaccharide standards at equal molar concentration, as defined by their UV absorption at 232 nm)

The Equimolar Stock Solution prepared here consists of 100 µM of each disaccharide


Note: The concentrations and volumes described here apply only to our current Disaccharide Stocks, which have been corrected for their UV absorption at 232 nm to provide UV-corrected concentrations (as described in the preceding sections of this protocol).

If using different Disaccharide Stocks, the concentrations and volumes will need to be updated.

HS 100 μM Equimolar Stock Solution


Note

1. Use the formula to calculate the volume of each Disaccharide Stock required to make a single Equimolar Stock Solution (consisting of 100 µM of each HS disaccharide). Here, 100 µL will be prepared.


rearranged provides:

where:
C1 = Starting UV-corrected concentration (mM)
V1 = Volume of stock needed ( μL)
C2 = Desired concentration (mM)
V2 = Desired total volume (μL)

-----------------------------------------------------------------------------------------------------

For example for our current HD001 Disaccharide Stock*:




*this applies only to our current HD001 Disaccharide Stock, which was determined above to have a UV-Corrected concentration of 3.976 mM (as described above). If using different Disaccharide Stocks, the concentrations and volumes will need to be updated.


2. Repeat the above for each disaccharide


3. Sum the volumes of all Disaccharide Stocks


4. Calculate the volume of water required to add to make the volume up to 100 μL (= 100 - summed volume of all Disaccharide Stocks)

-----------------------------------------------------------------------------------------------------

For example, for our current HS Disaccharide Stocks**:

ABCDE
Analyte nameUV-corrected concentration of Disaccharide Stock (mM)Volume of Disaccharide Stock required (V1, uL)Summed voilume of all disaccharides (uL)Volume of ddH2O required (uL)
HD0013.9762.537.962.1
HD0021.7765.6
HD0031.6855.9
HD0041.9915.0
HD0051.9585.1
HD0064.0212.5
HD0071.7855.6
HD0081.7975.6
**this applies only to our current Disaccharide Stocks. If using different Disaccharide Stocks, the concentrations and volumes will need to be updated.





Prepare 100 µL of a single Equimolar Stock Solution of each HS disaccharide:

  • Thaw an aliquot of each HS Disaccharide Stock ( for Disaccharide Stock preparation )
  • To a single microfuge tube, add the volumes of each Disaccharide Stock calculated in column C of the table above
  • To the same tube, add the required amount of water to make the total volume 100 μL, here 61.2 μL (see note above for calculations)
  • Mix well
  • Store at4 °C for around one month‡
  • Aliquots can also be made and stored at -20oC for up to 6 months‡. Avoid repeat freeze-thaw cycles.

‡ Longer stability may be possible but has not been tested at the time of publication of this protocol.



CS/DS/HA 100 μM Equimolar Stock Solution



Note
1. Use the formula to calculate the volume of each Disaccharide Stock required to make a single Equimolar Stock Solution (consisting of 100 µM of each CS/DS/HA disaccharide). Here, 100 µL will be prepared.


rearranged provides:

where:
C1 = Starting UV-corrected concentration (mM)
V1 = Volume of stock needed ( μL)
C2 = Desired concentration (mM)
V2 = Desired total volume (μL)

-----------------------------------------------------------------------------------------------------

For example for our current CD001 Disaccharide Stock*:




*this applies only to our current CD001 Disaccharide Stock, which was determined above to have a UV-Corrected concentration of 2.494 mM (as described above). If using different Disaccharide Stocks, the concentrations and volumes will need to be updated.


2. Repeat the above for each disaccharide


3. Sum the volumes of all Disaccharide Stocks


4. Calculate the volume of water required to add to make the volume up to 100 μL (= 100 - summed volume of all Disaccharide Stocks)

-----------------------------------------------------------------------------------------------------

For example, for our current CS/DS/HA Disaccharide Stocks**:


ABCDE
Analyte nameUV-corrected concentration of Disaccharide Stock (mM)Volume of Disaccharide Stock required (V1, uL)Summed voilume of all disaccharides (uL)Volume of ddH2O required (uL)
CD0012.4944.078.621.4
CD0022.8293.5
CD0032.1434.7
CD0040.65815.2
CD0050.56917.6
CD0060.81512.3
CD0080.69014.5
HA021.4486.9
**this applies only to our current Disaccharide Stocks. If using different Disaccharide Stocks, the concentrations and volumes will need to be updated.




Prepare 100 µL of a single Equimolar Stock Solution of each CS/DS/HA disaccharide:

  • Thaw an aliquot of each CS/DS/HA Disaccharide Stock ( for Disaccharide Stock preparation )
  • To a single microfuge tube, add the volumes of each Disaccharide Stock calculated in column C of the table above (see note above for calculations)
  • To the same tube, add the required amount of water to make the total volume 100 μL, here 21.4 μL (see note above for calculations)
  • Mix well
  • Store at 4 oC for around one month‡
  • Aliquots can also be made and stored at -20oC for up to 6 months‡. Avoid repeat freeze-thaw cycles.

‡ Longer stability may be possible but has not been tested at the time of publication of this protocol.




Preparation of Equimolar Stock Dilution Series

Note
The 100 μM Equimolar Stock Solution can used to prepare a dilution series for (a) generating a calibration curve of standards and/or (b) assessing linearity of responses in the subsequent HILIC-MS/MS method.

For example, prepare a dilution series using the 100 μM Equimolar Stock Solution as follows:

ABCD
Equimolar Stock Solution Concentration (uM)Equimolar Stock Solution usedVolume of Equimolar Stock Solution to add (uL)Volume of ddH2O to add (uL)
100n/a n/an/a
10100 uM1090
110 uM1090
0.11 uM1090
For each row in the table above, prepare the Equimolar Stock Solution as follows:
  • Label microfuge tubes according to column A
  • Use the Equimolar Stock Solution from column B, to add the volume in column C
  • Add the volume of ddH2O in column D
  • Mix well

For example for the preparation of the 1 μM Equimolar Stock Solution (row 4 of the table above):
  • Add 10 µL 10 μM Equimolar Stock Solution
  • Add 90 µL ddH2O
  • Mix well