Feb 09, 2024
Analysis of glycosphingolipids from cell lines
Forked from Analysis of glycosphingolipids from human plasma
- David A Priestman1,2,
- Yuzhe Weng1,
- Marya Sabir1,
- Reuben Bush1,
- Kerri-Lee Wallom1,
- María Fernández-Suárez1,
- Frances M Platt1,2,3,4,
- Danielle te Vruchte5
- 1Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, United Kingdom;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
- 3Royal Society Wolfson Research Merit Award Holder;
- 4Wellcome Trust Investigator in Science.;
- 5University of Oxford
Protocol Citation: David A Priestman, Yuzhe Weng, Marya Sabir, Reuben Bush, Kerri-Lee Wallom, María Fernández-Suárez, Frances M Platt, Danielle te Vruchte 2024. Analysis of glycosphingolipids from cell lines. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9pn14g3e/v1
Manuscript citation:
Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling DCA Neville, V Coquard, DA Priestman, DJM te Vruchte, DJ Sillence, Raymond A Dwek, Frances M Platt, Terry D Butters Analytical biochemistry 331 (2), 275-282, (2004). doi: 10.1016/j.ab.2004.03.051.
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: Working
We use this protocol and it's working
Created: January 25, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94146
Keywords: Glycosphingolipids, Glucosyl Ceramide, HPLC, Oligosaccharide Analysis, ASAPCRN, cellular glycosphingolipid, glycosphingolipid, ceramide glycanase enzyme, oligosaccharide, reproducible molar quantities of individual gsl species, individual gsl species, cell line sample, reproducible quantitation of gsl, fluorescent compound anthranilic acid, liquid chromatography, labelled chitotriose calibration standard, cell lines interest in the role, performance liquid chromatography, cell lines interest, present in mammalian cell, chitotriose calibration standard, gsl, mammalian cell
Funders Acknowledgements:
The Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Aligning Science Across Parkinson’s (ASAP) Initiative
Grant ID: ASAP-000478
Abstract
Interest in the role of cellular glycosphingolipids (GSLs) in health and disease led to us developing a sensitive method to analyse the full complement of GSL structures present in mammalian cells, fluids and tissues. The original qualitative method we developed was published in 2004 and measured the oligosaccharides selectively released from glycosphingolipids using a ceramide glycanase enzyme derived from the medicinal leech. We have now updated and refined this protocol with the focus on achieving sensitive and reproducible quantitation of GSLs in cell line samples. The method uses the fluorescent compound anthranilic acid (2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labelling procedure is rapid, selective, and easy to perform. With the inclusion of a 2AA-labelled chitotriose calibration standard, it is possible to obtain accurate and reproducible molar quantities of individual GSL species.
Guidelines
This protocol requires the use of some hazardous materials. As such, users must be appropriately trained and hazardous materials stored, used, and disposed of in accordance with your institution’s health and safety policies, and approved laboratory policies, risk assessments and codes of practice.
Materials
Acetonitrile 1.00030
Gradient grade for liquid chromatography LiChrosolv® Reag. Ph Eur
Anthranilic acid
A89855 Sigma-Aldrich
Reagent grade, ≥98%
Bicinchoninic Acid solution
Boric acid
ReagentPlus®, ≥99.5%
B0252 Sigma-Aldrich
Chloroform
Suitable for HPLC, ≥99.8%, amylene stabilized
34854-M Sigma-Aldrich
Copper(II) sulfate solution
Discovery® DPA-6S SPE Tube
Gibco™ DPBS, no calcium, no magnesium
14190094
Glycosphingolipid standards
Kinesis SPE Columns: TELOS® C18(EC) 100mg/1ml SPE Columns
Ludger - BioQuant 2AA Labelled Chitotriose Standard
Cat. #: BQ-CAA-CHI-01 Batch: B37I-02
Ludger - 2-AA Labeled Glucose Homopolymer Ladder
Cat. # CAA-GHP-30
Methanol
34860 Sigma-Aldrich
Suitable for HPLC, ≥99.9%
rEGCase I: recombinant Endoglycoceramidase custom-synthesized by GenScript
Sarstedt 1.5 ml Micro tubes with screw cap and seal
Product reference number 72.692
Sodium acetate trihydrate
S1304 Sigma-Aldrich
Meets USP testing specifications
Sodium cyanoborohydride
Reagent grade, 95%
156159 Sigma-Aldrich
TSKgel® Amide-80 HPLC Column
Safety warnings
This protocol requires the use of some hazardous solvents, reagents and chemicals. Refer to the Safety Data Sheets (SDS) provided by supplier and applicable Control of Substances Harmful to Health (COSHH). The correct personal protective equipment must be worn, and incidents reported in line with your institution’s policy and procedures.
Before start
Check that you have the required reagents, solvents, chemicals, equipment and PPE.
GSL preparation from cell lines
Lyse and homogenise cell pellet (≥ 1 ⋅ 106 cells) in 200 µL ddH2O through three cycles of freeze-thawing with vortexing after each cycle.
Measure protein concentration using the Bicinchoninic acid assay (BCA) method in triplicate at 4 fold dilution (repeat if triplicate values are not in agreement).
Using determined protein concentrations, prepare sample as 200 µg protein in 200 µL ddH2O in a 1.5 ml screw-cap tube.
Add 0.8 mL of chloroform/methanol (1:2, v/v) to give (C/M/W 4:8:3 final).
Safety information
Chloroform and methanol are very toxic, and methanol is flammable. Refer to the Safety Data Sheets.
Leave Overnight at 4 °C .
Vortex.
Centrifuge at 16,000 x g for 00:10:00 at Room temperature .
10m
Transfer supernatant (about 1 mL ) to new tube and separate into two phases: by adding 0.2 mL PBS and then 0.2 mL chloroform.
Vortex.
Centrifuge at 16,000 x g for 00:10:00 at room temperature.
10m
Remove very carefully the lower phase to a new tube and retain the upper phase.
Dry down the lower phase under a stream of (oxygen-free) nitrogen in heating block (42 °C ).
When dry, re-suspend the lower phase in 20 µL chloroform/methanol (1:3).
Add upper phase to lower phase and vortex.
Pre-equilibrate C18 columns (telos, Kinesis, UK) with 4 x 1 mL methanol and 3 x 1 mL deionised water.
Load lower/upper phase mix onto column, let drip through gravity flow.
Rinse sample tube with 1 mL water, apply to column to wash.
Wash column with 3 x 1 mL water.
Elute GSLs into a new tube with:
2 mL chloroform/methanol (98:2). Push through first 0.5 mL . You can use syringe with adapter that fits into top of column.
2 mL chloroform/methanol (1:3).
1 mL methanol.
Vortex and leave Overnight at 4 °C or carry on to enzymatic digestion.
GSL digestion with EGC’ase I
16h
Vortex (5 mL , C18) and dry down samples under a stream of nitrogen in heating block (42 °C ).
When about 150 µL sample remaining, transfer to 1.5 mL screw-cap tube.
Rinse sample tube with 200 µL C:M 2:1, vortex and combine with the rest of the sample in the screw-cap tube.
Rinse sample tube with 200 µL chloroform, vortex and combine with the rest of the sample in the screw-cap tube.
Dry down, under a slow stream of nitrogen in heating block (42 °C ).
Re-suspend in 50 µL C:M 2:1, vortex, dry down under a very slow stream of nitrogen.
Note
NB It is essential that ALL the sample is dried in the bottom of the tube.
Add 90 µL enzyme/buffer to each sample and vortex:
rEGCase : 5 µL enzyme (stored in freezer) plus 85 µL buffer per sample.
Note
rEGCase buffer (stored at 4 °C ): 0.6 % (v/v) Triton in 50 millimolar (mM) sodium acetate pH 5.2.
rEGCase buffer (stored at 4 °C ): 0.6 % Triton in 50 millimolar (mM) sodium acetate pH 5.2.
Incubate at 37 °C for 16:00:00 .
Note
rEGCaseI: recombinant Endoglycoceramidase I was custom synthesised by GenScript.
16h
2AA labelling of glycans released from GSLs
1h 30m
Note
Labelling mix: 30 mg/mL 2AA and 45 mg/mL sodium cyanoborohydride, in labelling buffer
Labelling buffer: 4% sodium acetate and 2% boric acid in methanol
Dissolve 2AA in labelling buffer first with vortexing, then add the sodium cyanoborohydride and vortex.
Safety information
Sodium cyanoborohydride is very toxic. Refer to the Safety Data Sheet.
Add 310 µL labelling mix to the 90 µL sample (digest) in 1.5 mL screw-cap tube.
Incubate in oven at 80 °C for 01:00:00 , vortexing at 00:30:00 .
1h 30m
Allow to cool to Room temperature .
Add 1 mL acetonitrile:water (97:3).
Transfer from screw-cap tube to 15 mL tubes.
Rinse screw-cap tube with 2 x 1 mL acetonitrile:water (97:3) and add to 15 mL tube.
Pre-equilibrate 50 mg Discovery® DPA-6S SPE Tube (supplied by Sigma-Aldrich) with:
1 mL acetonitrile
2 x 1 mL water
2 x 1 mL acetonitrile
Apply samples (3.4 mL ) to equilibrated DPA-6S columns, let drip through gravity flow.
Wash columns with acetonitrile:water (95:5) → add 1 mL into the 15 mL tubes to wash and add to columns, and then add 3 x 1 mL acetonitrile:water (95:5) directly onto the columns.
Elute with 600 µL water into new tubes.
Add 60 µL :140 µL (sample:acetonitrile) to HPLC vials and vortex.
For HPLC, inject 5 µL -50 µL .
DPA-6S eluate is stable stored at -20 °C .
HPLC protocol
1h
Purified 2AA-labeled oligosaccharides are separated and quantified by normal-phase high-performance liquid chromatography (NP-HPLC) as previously described (Neville et al., 2004, see the reference below).
Note
DCA Neville, V Coquard, DA Priestman, DJM te Vruchte, DJ Sillence, Raymond A Dwek, Frances M Platt, Terry D Butters
Analytical biochemistry 331 (2), 275-282, (2004).
doi: 10.1016/j.ab.2004.03.051.
The HPLC system consists of a Waters Alliance 2695 separations module and an in-line Waters 2475 multi λ-fluorescence detector set at Ex λ360 nm and Em λ425 nm.
The solid phase used is a 4.6 × 250 mm TSK gel-Amide 80 column maintained at 30 °C (Anachem, Luton, UK).
The chromatographic flow rate is 0.8 mL/min, and run time was 60 min. The total run time is 01:00:00 .
Gradient conditions for Normal Phase HPLC. All chromatography was controlled and data were collected and processed using Waters Empower software.
1h
The mobile phases are acetonitrile (solvent A), de-ionised water (solvent B) and 100 millimolar (mM) ammonium acetate, 3.85 (solvent C).
Starting conditions are 71.6% solvent A. 8.4% solvent B and 20% solvent C at 0.8 mL/min constant flow-rate for 6 mins. 00:06:00
6m
The gradient is developed from 6 min to 35 min by increasing solvent B from 8.4% to 27.2% with concomitant decrease in solvent A from 71.6% to 52.8 %. 00:29:00
29m
From 35 to 37 min solvent B is increased to 57% and solvent A reduced to 23%, then maintained for 2 min up until 39 min. 00:04:00
4m
Between 39 and 41 min the solvent ratios are returned to the starting conditions and then maintained for 1 min. 00:03:00
3m
At 42 mins the flow-rate is increased to 1.2 mL/min and maintained for 12 min up to 54 min. 00:12:00
12m
Between 54 and 60 min the flow-rate is gradually returned to 0.8 mL/min. 00:06:00
6m
Individual GSL species are identified by their glucose unit values (GUs), calculated with the HPLC Empower software using a homopolymer dextran ladder (Fig. 1).
To help identify GSL peaks in the samples, compare the GUs of the peaks in the samples with those in a GSL standard mix.
Prepare a mixture of commercially available authentic GSLs in a screw-cap tube:
50 µL Matreya Neutral GSL mix Cat No 1505 (Glc-Cer, Lac-Cer, Gb3, Gb4) 1.0 mg/ml
100 µL Matreya Ganglioside mix Cat No 1510 (Lac-Cer, GM3, GD3) 0.5 mg/ml
100 µL Matreya Ganglioside mix Cat No 1511 (GA1, GM1a, GD1a, GD1b,GT1b) 0.5 mg/ml
30 µL Matreya GM2 Ganglioside Cat No 1502 (GM2) 0.5 mg/ml
30 µL Sigma Asialo-GM2 Cat No G9398 (GA2) 1.0 mg/ml
Dry down the mixture under nitrogen and then digest, label and clean up as done for the samples in steps 25-41.
Aliquot and store at-20 °C .
In order to calculate molar quantities from integrated peaks in the chromatogram, inject a calibration standard containing 2.5 pmol 2AA-labelled chitotriose (Ludger) with each sample set (not shown).
Figures 1- 3: HPLC profile and sugar structures for GSLs in cell lines together with the GSL biosynthetic pathway
Figure 1 HPLC profiles for GSL standards and 2AA-labelled homopolymer dextran ladder. Glucose Units were calculated using the retention times of a 2AA-labelled homopolymer dextran ladder and are shown for each of the GSL standard peaks.
Figure 2 GSL biosynthesis. Biosynthetic enzymes genes are indicated in the blue grid. Ganglioside names are abbreviated according to Svennerholm [1]. [1] L. Svennerholm, Designation and schematic structure of gangliosides and allied glycosphingolipids, Prog Brain Res 101 (1994) XI-XIV.
Figure 3 Typical HPLC profile for 2AA-labelled glycans released from RAW (top panel), HEK (middle panel), and human fibroblasts (bottom panel) glycosphingolipids with sugar structures indicated for each peak. In order to calculate values for GSL concentrations, the peaks are integrated and compared with a quantitative standard of known concentration.
Note
Figures created with BioRender.com