Apr 28, 2026
HILIC-MS/MS data acquisition and processing of label-free GAG disaccharides
- H Davies-Strickleton1,2,
- George Taylor2,
- 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, George Taylor, James Allsey, Douglas Dyer, David Knight 2026. HILIC-MS/MS data acquisition and processing of label-free GAG disaccharides. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld6keog5b/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 28, 2025
Last Modified: April 28, 2026
Protocol Integer ID: 233735
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 of label, gag disaccharide preparation sample preparation, relative quantification of gag disaccharide amount, free gag disaccharides from biological tissue, gag disaccharide amount, gag disaccharide preparation, gag disaccharide standard, free gag disaccharide hilic, gag disaccharide, free gag disaccharide, disaccharide amount, free gag disaccharides this protocol, disaccharide composition, disaccharide, ms analysis from biological tissue, equimolar disaccharide stock, summation of disaccharide amount, relative ratio of disaccharide, sample preparation of label, biological tissues equimolar stock solution preparation, total gag amount, samples for analysis hilic, sample preparation, ms data acquisition, step guide for hilic, ms analysis, data processing of label, ms acquisition
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 HILIC-MS/MS acquisition and data processing of label-free GAG disaccharides. This enables relative quantification of GAG disaccharide amounts, total GAG amounts (by summation of disaccharide amounts) and disaccharide composition (relative ratio of disaccharides compared to total GAG).
This is the last protocol in a collection, which documents the details of all steps required for label-free GAG disaccharide HILIC-MS/MS analysis from biological tissues:
- HILIC-MS/MS data acquisition and processing
Prior to this protocol, biological material was disrupted and GAG disaccharides generated (according to protocols 1-2), Equimolar Disaccharide Stocks were prepared (protocol 3), and both Samples and Standards were vialled (protocol 4).
Materials
Materials
- The reagents listed are those used in the development of this protocol.
- Alternatives are also available and should be LC-MS grade
LC-MS instrumentation
- This protocol was developed on a Shimadzu Nexera LC 30 AD coupled to a SCIEX ZenoTOF 7600 mass spectrometer
- The column used was an InfinityLab Poroshell 120 HILIC-Z, 2.1 x 150 mm, 2.7 µm Column (Agilent, 683775-924), coupled to an InfinityLab Poroshell 120 HILIC-Z, 2.1 mm, 2.7 µm, UHPLC guard (Agilent, 821725-947)
Protocol materials
Ammonium acetateVWR International (Avantor)Catalog #84885.180
Acetonitrile LC-MS GradeFisher ScientificCatalog #A/0638/15
Troubleshooting
Problem
Unexpected peak retention or loss of signal for some injections
Solution
Examine the LC pressure trace - they should overlap for all injections of a batch
If they do not there could be a leak in the LC system, or the guard column or column may need replacing
Safety warnings
Repeat injections
It is recommended that repeat injections, if needed, be made by preparing multiple vials, rather than by performing multiple injections from a single vial
- This is based on observations of signal depletion with repeat injections from a single vial, thought to be due to potential nucleation/precipitation induced by the injector needle coming into contact with the sample.
- Injector precision is not impacted when repeat injections are made from individual vials.
Buffer preparation
250 mM Ammonium Acetate Stock
- Weigh 19.27 g Ammonium acetateVWR International (Avantor)Catalog #84885.180
- Add to 1 L ddH2O in a duran bottle
- Mix well
- Can be stored for 1 year at 4 °C
Note
Exact weights and volumes can be adjusted using the formula below.
Weigh approximately 19.27 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) = 0.25
MW = molecular weight = 77.08
Example
If 20 g ammonium acetate was weighed then the volume required would be:
20/(0.25*77.08) = 1.037 L
-----------------------------------------------------------------------------------------------------
Mobile Phase A (25 mM ammonium acetate in water)
Note
Mobile Phase A is prepared by combining:
- 9 parts water
- 1 parts 250 mM Ammonium Acetate Stock (1 in 10 dilution to give final concentration of 25 mM)
- Total: 10 parts
An example preparation for 500 mL is given below. Should a higher volume be needed, multiply all component volumes accordingly (e.g. for 1000 mL, multiply all the volumes below by 2)
For example to prepare 500 mL in a duran bottle:
- Add 450 mL ddH2O
- Add 50 mL 250 mM Ammonium Acetate Stock for buffer preparation
- Mix
- Can be used for up to 1 month
Mobile Phase B (25 mM ammonium acetate in 85% acetonitrile)
Note
Mobile Phase B is prepared by combining:
- 85 parts acetonitrile
- 10 parts 250 mM Ammonium Acetate Stock (1 in 10 dilution to give final concentration of 25 mM)
- 5 parts water
- Total: 100 parts
An example preparation for 1 L is given below. Should a lower volume be needed, divide all component volumes accordingly (e.g. for 500 mL, divide all the volumes below by 2)
For example to prepare 1000 mL in a duran bottle:
- Add 850 mL Acetonitrile LC-MS GradeFisher ScientificCatalog #A/0638/15
- Add 100 mL 250 mM Ammonium Acetate Stock for stock preparation
- Add 50 mL ddH2O
- Mix and allow to reach room temperature
- Can be used for up to 3 months
Wash (80% acetonitrile)
Note
The Wash is prepared by combining:
- 4 parts acetonitrile
- 1 part water
- Total: 5 parts
An example preparation for 300 mL is given below. Should a higher volume be needed, multiply all component volumes accordingly (e.g. for 600 mL, multiply all the volumes below by 2)
For example to make 300 mL in a duran bottle:
- Add 240 mL Acetonitrile LC-MS GradeFisher ScientificCatalog #A/0638/15
- Add 60 mL ddH2O
- Mix and allow to reach room temperature
- Can be used for up to 3 months
Storage Buffer (50% acetonitrile, 5 mM ammonium acetate)
Note
The Storage Buffer is prepared by combining:
- 25 parts acetonitrile
- 24 parts water
- 1 parts 250 mM Ammonium Acetate Stock (1 in 50 dilution to give final concentration of 5 mM)
- Total: 50 parts
An example preparation for 100 mL is given below. Should a higher volume be needed, multiply all component volumes accordingly (e.g. for 200 mL, multiply all the volumes below by 2)
For example to make 100 mL in a duran bottle:
- Add 50 mL Acetonitrile LC-MS GradeFisher ScientificCatalog #A/0638/15
- Add48 mL ddH2O
- Add 2 mL 250 mM ammonium acetate for stock preparation
- Mix and allow to reach room temperature
- Can be used for up to 3 months
Heparan Sulphate (HS) data acquisition method setup
In the LC-MS software, create the LC-MS acquisition method parameters for the analysis of HS disaccharides
Note
Create acquisition methods using the LC-MS conditions detailed here.
Note: exact LC conditions may need to be optimised for isomer separation (e.g. via test injections as described later).
- This is based on the observation that new HILIC-Z columns, and columns that had been stored, required LC gradients to be adjusted by up to +/- 5% B.
MS settings (CE and/or DP) can also be adjusted to ensure satisfactory fragmentation.
Typical LC conditions for HS disaccharides
| A | B | C | |
| LC settings | |||
| Cooler Temperature (oC): | 5 | ||
| Injection Volume (uL) | 5 | ||
| Oven temperature (oC): | 50 | ||
| LC gradient | |||
| Time [min] | Flow [mL/min] | B.Conc [%] | |
| 0 | 0.25 | 94 | |
| 3 | 0.25 | 94 | |
| 25 | 0.25 | 90 | |
| 25.1 | 0.25 | 5 | |
| 27 | 0.25 | 5 | |
| 27.1 | 0.25 | 94 | |
| 30.1 | 0.25 | 94 | |
Note
Sample injection volume
Different injection volumes have been tested and 5 μL provides good signal with good isomer separation. Isomer resolution becomes poorer with higher injection volume.
Typical MS/MS parameters for HS disaccharides
| A | B | |
| MS Source parameters | ||
| Method duration (minutes): | 30 | |
| Source name: | OptiFlow 50-200µL Micro/MicroCal | |
| Curtain gas: | 30 | |
| CAD gas: | 7 | |
| Ion source gas 1 (psi): | 50 | |
| Ion source gas 2 (psi): | 70 | |
| Temperature (°C): | 250 | |
| Scan type: | MRMhr | |
| Polarity: | Negative | |
| Spray voltage (V): | -4500 | |
| A | B | C | D | E | F | G | |
| TOF-MS/MS parameters | |||||||
| Compound ID | Precursor Ion | TOF Start Mass | TOF Stop Mass | Accumulation Time | Declustering potential | Collision energy | |
| HD001 | 287.4784 | 60 | 600 | 0.1 | -20 | -12 | |
| HD002 & HD004 | 247.5001 | 60 | 600 | 0.1 | -40 | -20 | |
| HD003 | 268.5049 | 50 | 500 | 0.1 | -80 | -20 | |
| HD005 | 416.0514 | 50 | 500 | 0.1 | -60 | -30 | |
| HD006 | 378.1048 | 40 | 400 | 0.1 | -80 | -20 | |
| HD007 & HD008 | 458.0617 | 50 | 500 | 0.1 | -60 | -35 | |
| Internal standard | 552.0336 | 60 | 600 | 0.1 | -40 | -20 | |
A TOF-MS scan was also included using the following parameters:
- TOF start and stop mass: 50-1000 Da
- Accumulation time: 0.8s
- Declustering potential: -40 V
- Collision energy: -5 V
Chondroitin Sulphate/ Dermatan Sulphate/ Hyaluronan (CS/DS/HA) data acquisition method setup
In the LC-MS software, create the LC-MS acquisition method parameters for the analysis of CS/DS/HA disaccharides
Note
Create acquisition methods using the LC-MS conditions detailed here.
Note: exact LC conditions may need to be optimised for isomer separation (e.g. via test injections as described later).
- This is based on the observation that new HILIC-Z columns, and columns that had been stored, required LC gradients to be adjusted by up to +/- 5% B.
MS settings (CE and/or DP) can also be adjusted to ensure satisfactory fragmentation.
Typical LC conditions for CS/DS/HA disaccharides
| A | B | C | |
| LC settings | |||
| Cooler Temperature (oC): | 5 | ||
| Injection Volume (uL) | 5 | ||
| Oven temperature (oC): | 50 | ||
| LC gradient | |||
| Time [min] | Flow [mL/min] | B.Conc [%] | |
| 0 | 0.25 | 98 | |
| 16 | 0.25 | 98 | |
| 24 | 0.25 | 87 | |
| 26 | 0.25 | 87 | |
| 26.2 | 0.25 | 5 | |
| 30 | 0.25 | 5 | |
| 30.1 | 0.25 | 98 | |
| 35.1 | 0.25 | 98 | |
Note
Sample injection volume
Different injection volumes have been tested and 5 μL provides good signal with good isomer separation. Isomer resolution becomes poorer with higher injection volume.
Typical MS/MS parameters for CS/DS/HA disaccharides
| A | B | |
| MS Source parameters | ||
| Method duration (minutes): | 35 | |
| Source name: | OptiFlow 50-200µL Micro/MicroCal | |
| Curtain gas: | 30 | |
| CAD gas: | 7 | |
| Ion source gas 1 (psi): | 50 | |
| Ion source gas 2 (psi): | 70 | |
| Temperature (°C): | 250 | |
| Scan type: | MRMhr | |
| Polarity: | Negative | |
| Spray voltage (V): | -4500 | |
| A | B | C | D | E | F | G | |
| TOF-MS/MS parameters | |||||||
| Compound ID | Precursor Ion | TOF Start Mass | TOF Stop Mass | Accumulation Time | Declustering potential | Collision energy | |
| HA02 & CD001 | 378.1041 | 40 | 400 | 0.1 | -30 | -18 | |
| CD002 & CD003 & CD008 | 458.0614 | 50 | 500 | 0.1 | -40 | -33 | |
| CD004 & CD005 & CD006 | 268.5053 | 60 | 600 | 0.1 | -60 | -18 | |
| Internal standard | 552.0336 | 60 | 600 | 0.1 | -40 | -20 | |
A TOF-MS scan was also included using the following parameters:
- TOF start and stop mass: 50-1000 Da
- Accumulation time: 0.8s
- Declustering potential: -40 V
- Collision energy: -5 V
LC-MS system operation and optimisation
Degas solvents:
- Mobile Phase A
- Mobile Phase B
- Wash
Purge the LC system with the Mobile Phases and Wash solvent
Install the HILIC-Z column and guard
Equipment
P120 HILIC-Z, 2,1×150 mm, 2,7 µm
NAME
HPLC Column
TYPE
Agilent Technologies
BRAND
673775-924
SKU
Equipment
InfinityLab Poroshell 120 HILIC Guard
NAME
Guard column
TYPE
Agilent
BRAND
821725-947
SKU
Equilibrate the column with gradient starting conditions
- For HS analysis for LC-MS method parameters
- CS/DS/HA analysis for LC-MS method parameters
Set off test injections of an Equimolar Disaccharide Standard
- A high concentration such as 5000 nM is recommended for test injections
- Adjust LC-MS/MS conditions if necessary to achieve isomer separation and satisfactory fragmentation
Perform test injections for the other GAG type and acquisition method.
HILIC-MS/MS batch data acquisition
Ensure the vials from the preceding protocol have been placed in the autosampler 90 minutes prior to injection
- This steps ensures the samples reach the temperature of the autosampler before injection. It has been observed that the intensity of the disaccharides increases as the temperature reaches 5 oC.
Note
Repeat injections
It is recommended that repeat injections, if needed, be made by preparing multiple vials, rather than by performing multiple injections from a single vial
- This is based on observations of signal depletion with repeat injections from a single vial, thought to be due to potential nucleation/precipitation induced by the injector needle coming into contact with the sample.
- Injector precision was not impacted when repeat injections were made from individual vials.
Set off a batch of injections for data acquisition by the HILIC-MS/MS method
For each GAG type, the following should be analysed:
- Blank Diluent
- Blank Internal Standard
- Equimolar Disaccharide Standard: 50 nM
- Positive Control Sample
- Test Samples
Optional:
- Equimolar Disaccharide Linearity Standards: 5, 500 and 5000 nM
Note
The rationale for their analysis is described here:
Blank Diluent
- Should be free from analyte responses
Blank Internal Standard
- Should be positive for the Internal Standard analyte, and be free from analyte responses
Equimolar Disaccharide Standard: 50 nM
- The purpose of this 50 nM Equimolar Disaccharide Standard is to correct for disaccharide specific variability in analysis and to provide a single point calibration for the approximation of disaccharide amount (semi-quantification).
Positive Control Sample
- The Positive Control Sample should provide a consistent disaccharide profile with previous data
Test Samples
- The Test Samples are analysed to determine their relative amounts of GAG disaccharides, total GAG, and disaccharide composition
Optional Equimolar Disaccharide Linearity Standards: 5, 500 and 5000 nM
- Additional Equimolar Disaccharide Standards at different concentrations can be used for (a) test injections to optimise LC-MS conditions prior to analysis and (b) to confirm analyte linearity
After all batches are acquired, store the column and guard in Storage Buffer ( for buffer preparation)
HILIC-MS/MS data processing
Integrate peaks across all injections of an analytical batch
Here, SCIEX OS (V3.0) is used to integrate peak areas
- Use the peak-to-peak signal-to-noise algorithm
- Peak areas should have a signal-to-noise value ≥ 3, or they are deemed below the criteria for detection
HS disaccharide peak integration
Note
The fragment ions to be used for quantification were selected based on their specificity at the selected retention time and their positive annotation in GlycoWorkbench [reference 1].
For many disaccharides there are multiple ion transitions that can be summed for added sensitivity.
List of ion transitions (precursors and fragment ions) for HS disaccharide analysis:
| A | B | C | D | E | |
| Structure | Analyte Name | Relative Retention Time* | Precursor (Q1) Mass (Da) | Fragment (Q3) Mass (Da) | |
| ∆UA-2S GlcNCOEt-6S | Internal standard (IS) | 1.00 | 552.0336 | 472.0781 | |
| ΔUA,2S-GlcNS,6S | HD001 | 2.13 | 287.4784 | 96.9599 | |
| 287.4784 | 247.5 | ||||
| 287.4784 | 137.9865 | ||||
| 287.4784 | 357.0129 | ||||
| 287.4784 | 157.0141 | ||||
| 287.4784 | 287.4784 | ||||
| ΔUA,2S-GlcNS | HD002 | 1.46 | 247.5 | 96.96 | |
| 247.5 | 247.5 | ||||
| 247.5 | 137.9866 | ||||
| 247.5 | 157.0143 | ||||
| 247.5 | 357.0139 | ||||
| ΔUA,2S-GlcNAc,6S | HD003 | 1.25 | 268.5049 | 96.9602 | |
| 268.5049 | 157.0143 | ||||
| 268.5049 | 138.9711 | ||||
| 268.5049 | 370.046 | ||||
| 268.5049 | 268.5049 | ||||
| ΔUA-GlcNS,6S | HD004 | 1.61 | 247.5 | 300.0404 | |
| 247.5 | 247.5 | ||||
| 247.5 | 258.0303 | ||||
| 247.5 | 96.96 | ||||
| 247.5 | 157.0146 | ||||
| 247.5 | 137.9866 | ||||
| 247.5 | 357.0139 | ||||
| ΔUA-GlcNS | HD005 | 1.21 | 416.0514 | 137.9867 | |
| 416.0514 | 416.0514 | ||||
| 416.0514 | 175.0255 | ||||
| ΔUA-GlcNAc | HD006 | 0.76 | 378.1048 | 378.1048 | |
| 378.1048 | 175.0251 | ||||
| 378.1048 | 157.015 | ||||
| 378.1048 | 277.0567 | ||||
| ΔUA,2S-GlcNAc | HD007 | 0.85 | 458.0617 | 236.9712 | |
| ΔUA-GlcNAc,6S | HD008 | 0.92 | 458.0617 | 175.0256 | |
| 458.0617 | 357.0148 | ||||
| 458.0617 | 300.0403 |
*Exact values of relative retention time may vary with new columns
For the internal standard:
- Generate an extracted ion chromatogram of the ion transition listed in the table above (row 2)
- Integrate the peak found = internal standard peak area
For each disaccharide:
- Generate a summed◊ extracted ion chromatogram of the ion transitions listed in the table above
- Integrate the peak found at the relative retention time (relative to the internal standard retention time) = disaccharide peak area
- Calculate disaccharide peak area ratio = disaccharide peak area/ internal standard peak area
◊In the event of poor signal-to-noise, individual ion transitions listed in the table may examined in place of summed ions.
Peak acceptance criteria:
- Peaks should have signal to noise ≥3
Example data
Expected result
Peak integration of the internal standard (blue). The region used for calculating signal-to-noise is shown in grey.
Expected result
Example summed extracted ion chromatographs from a 50 nM Equimolar Disaccharide Standard of HS disaccharides:
- * denotes the analyte peak, other peaks are due to isomers
CS/DS/HA disaccharide peak integration
Note
The fragment ions to be used for quantification were selected based on their specificity at the selected retention time and their positive annotation in GlycoWorkbench [reference 1].
For many disaccharides there are multiple ion transitions that can be summed for added sensitivity.
List of ion transitions (precursors and fragment ions) for CS/DS/HA disaccharide analysis:
| A | B | C | D | E | |
| Structure | Analyte Name | Relative Retention Time* | Precursor (Q1) Mass (Da) | Fragment (Q3) Mass (Da) | |
| ∆UA-2S GlcNCOEt-6S | Internal standard (IS) | 1.00 | 552.0336 | 472.0781 | |
| ∆UA-(1–3)-GlcNAc | HA02 | 0.72 | 378.1041 | 175.0256 | |
| 378.1041 | 157.0146 | ||||
| 378.1041 | 378.1045 | ||||
| ∆UA-GalNAc | CD001 | 0.85 | 378.1041 | 175.0249 | |
| 378.1041 | 157.0143 | ||||
| 378.1041 | 378.1045 | ||||
| ∆UA-GalNAc,4S | CD002 | 1.04 | 458.0614 | 300.0399 | |
| 458.0614 | 342.0504 | ||||
| 458.0614 | 175.0247 | ||||
| 458.0614 | 157.0143 | ||||
| 458.0614 | 282.029 | ||||
| 458.0614 | 458.0614 | ||||
| ∆UA-GalNAc,6S | CD003 | 0.97 | 458.0614 | 282.0288 | |
| 458.0614 | 175.0248 | ||||
| ∆UA-GalNAc,4S,6S | CD004 | 1.15 | 268.5053 | 282.0294 | |
| 268.5053 | 175.0249 | ||||
| ∆UA,2S-GalNAc,4S | CD005 | 1.13 | 268.5053 | 300.0398 | |
| ∆UA,2S-GalNAc,6S | CD006 | 1.04 | 268.5053 | 300.0398 | |
| 268.5053 | 282.0294 | ||||
| 268.5053 | 157.0146 | ||||
| 268.5053 | 96.96 | ||||
| 268.5053 | 268.5053 | ||||
| ∆UA,2S-GalNAc | CD008 | 0.95 | 458.0614 | 236.971 |
*Exact values of relative retention time may vary with new columns
For the internal standard:
- Generate an extracted ion chromatogram of the ion transition listed in the table above (row 2)
- Integrate the peak found = internal standard peak area
For each disaccharide:
- Generate a summed◊ extracted ion chromatogram of the ion transitions listed in the table above
- Integrate the peak found at the relative retention time (relative to the internal standard retention time) = disaccharide peak area
- Calculate disaccharide peak area ratio = disaccharide peak area/ internal standard peak area
◊In the event of poor signal-to-noise, individual ion transitions listed in the table may examined in place of summed ions.
Peak acceptance criteria:
- Peaks should have signal to noise ≥3
Example data
Expected result
Peak integration of the internal standard (blue). The region used for calculating signal-to-noise is shown in grey.
Expected result
Example summed extracted ion chromatographs from a 50 nM Equimolar Disaccharide Standard of CS/DS/HA disaccharides.
- * denotes the analyte peak, other peaks are due to isomers
- ♦ denotes the HA02 peak and ‡ denotes the CD001
- Where two isomers have the same retention time, unique fragment ions provide unique analyte detection (eg CD004 vs CD005)
Initial review of results and acceptance criteria
Visual inspection of all peak integrations is recommended to ensure all peaks are correctly identified and well integrated
- For all injections, peaks detected should have signal to noise ≥3
Diluent and Blank Internal Standard detection criteria
Blank Diluent
- The Blank Diluent should be free from Disaccharide and Internal Standard responses
Blank Internal Standard
- Should be positive for the Internal Standard analyte, and be free from Disaccharide responses
However, it has been noted that a small amount of interference from the internal standard may be observed in the HD003 Disaccharide response
- To account for this, if there is interfering signal in either the Blank Diluent or Blank Internal Standard for a particular Disaccharide then the response of all other injections should only be accepted if it is ≥ 5x the response of the Blank Diluent or Blank Internal Standard.
Blank Internal Standard response across batch
Examine the internal standard peak area across the batch
- There should be fairly good consistency across the standards and the samples
- Some dropouts may occur and reflect either sample clean-up recovery efficiency, or LC-MS performance for the affected injections
- If regular dropouts occur, explore/troubleshoot further
50 nM Equimolar Disaccharide Standard
The 50 nM Equimolar Disaccharide Standard is used later for normalisation of Sample peak area ratios to a single calibration point
- Peak areas should have a signal-to-noise value ≥ 3
Optional: Linearity assessment
The 5, 500 and 5000 nM Equimolar Disaccharide Linearity Standards may also have been injected and can be used to gauge the linearity of the responses.
- If so, at least three concentrations (including 50 nM) of the Equimolar Disaccharide Standards should provide r2 ≥0.9 when peak area ratios are fitted with a linear regression
- Equimolar Disaccharide Standards should only be considered if the peak areas have a signal-to-noise value ≥ 3
Semi-quantification of disaccharides in samples
Note
For each disaccharide, peak area ratio in Sample is normalised to that of the Equimolar Disaccharide Standard, to correct for disaccharide specific variability in analysis and provide a single point calibration for the approximation of disaccharide amount in the extracted sample (semi-quantification).
The value is considered as semi-quantitative as (a) comparison is to a single calibration point and (b) full validation within each biological matrix has not been performed.
The semi-quantification presented here is appropriate for the relative quantification of disaccharide amounts, total GAG amounts (by summation of disaccharide amounts) and disaccharide composition (relative ratio of disaccharides compared to total GAG).
For all injections in a batch export all disaccharide peak area ratios to excel
Determine the amount of disaccharide in the extracted sample
- Use the following formula to determine the amount of disaccharide in the extracted sample
i.e.:
- Divide each the peak area ratio in Sample by the peak area ratio of the 50 nM Equimolar Disaccharide Standard
- Multiply by 20 pmol (this is the back calculated amount injected, as detailed in the note below)
Note
Back-calculated amount per sample
The 50 nM Equimolar Disaccharide Standard, when back-calculated provides the equivalent of 20 pmol of each disaccharide in the extracted sample, which is used to calculate an in-sample amount.
The calculations involved in the back-calculation are outlined here.
[1] Calculate the amount of disaccharide injected in the 50 nM Equimolar Disaccharide Standard reference (mol):
Where:
- Concentration = 50 nmol/L= 50x10-9 mol/L
- Volume = 5 μL = 5x10-6 L
Therefore:
- Amount injected in Standard (mol) = Concentration (mol/L) x Volume (L)
- mol = (50x10-9) x (5x10-6)
- mol = 250 x10-15 = 0.25 pmol
[2] Calculate the volume of the extracted sample that was injected:
- Sample volume in the injection vial = 3 μL
- Total volume in the injection vial = 60 μL
- Injection volume = 5 μL (as noted above)
Therefore:
- Volume of sample injected (μL) = 3 * (5/60) = 0.25 μL
[3] Combine the outputs from Equations [1] and [2] to calculate the amount of disaccharide present in the total extracted sample:
Where:
- Amount injected in Standard (mol) is obtained in Equation [1] = 0.25 pmol
- Volume of sample injected (uL) is obtained in Equation [2] = 0.25 μL
- Total volume of extracted sample = 20 μL (as per the protocol for reconstituting and vialling samples)
Therefore:
- Amount of disaccharide in total extracted sample (pmol) = 0.25 * (20/0.25) = 20 pmol
Sum all disaccharide amounts within each Test Sample and the Positive Control Sample
This provides a value for the total amount of HS or CS/DS per sample (pmol)
For HS:
- Sum all HS disaccharide amounts (pmol)
For CS/DS:
- Sum all CS/DS disaccharide amounts (pmol)
For HA:
- Only a single disaccharide is detected (HA02) and present in the HA structure, therefore the disaccharide amount = total HA amount (pmol)
Calculate % disaccharide composition of Test Samples and the Positive Control Sample
The % disaccharide composition is the relative ratio of disaccharides compared to total GAG. It is used to assess the sulphation profiles of GAGs isolated from biological material.
For HS:
- Divide the amount of each HS disaccharide (pmol) by the total sum of all HS disaccharides
- Multiply by 100 %
For CS/DS:
- Divide the amount of each CS/DS disaccharide (pmol) by the total sum of all CS/DS disaccharides
- Multiply by 100 %
Review the Positive Control Sample % disaccharide composition
- The % disaccharide composition of the Positive Control Sample analysed here should be consistent with that of previously analysed Positive Control Samples (of the same sample type)
Consider if additional normalisation is appropriate
- For comparison of GAG disaccharide and total GAG amounts across samples, the pmol/sample amounts can be normalised to amount of tissue, protein or cell counts.
Protocol references
[1] Ceroni A, Maass K, Geyer H, Geyer R, Dell A, Haslam SM. GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J Proteome Res. 2008 Apr;7(4):1650-9. doi: 10.1021/pr7008252. Epub 2008 Mar 1. PMID: 18311910.
Acknowledgements
We thank Rebecca Miller for guidance and advice regarding GAG disaccharide analysis.