TITLE: Analysis of glycosphingolipids from animal tissues
AUTHORS: David A Priestman, Marya Sabir, Reuben Bush, Danielle te Vruchte, Kerri-Lee Wallom, María E Fernández-Suárez, Frances M Platt

[DESCRIPTION]
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 animal tissues. 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.

[BEFORE_START]
Check that you have the required reagents, solvents, chemicals, equipment and PPE.


[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. 

[STEPS]
SECTION: GSL preparation from animal tissues
4. Add 0.8 mL of chloroform/methanol (1:2, v/v) to give (C/M/W 4:8:3 final).

 

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6. Leave  at 4 °C.

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7. Vortex.
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8. Centrifuge at 16,000 x g for 10 min at 4 Room temperature.

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9. 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.

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10. Vortex.
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11. Centrifuge at 16,000 x g for 10 min  at room temperature.

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12. Remove very carefully the lower phase to a new tube and retain the upper phase.

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13. Dry down the lower phase under a stream of (oxygen-free) nitrogen in heating block (42 °C).

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14. When dry, re-suspend the lower phase in 20 µL chloroform/methanol (1:3).

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15. Add upper phase to lower phase and vortex.
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16. Pre-equilibrate C18 columns (telos, Kinesis, UK) with 4 x 1 mL methanol and 3 x 1 mL deionised water.

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17. Load lower/upper phase mix onto column, let drip through gravity flow.
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18. Rinse sample tube with 1 mL water, apply to column to wash.

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19. Wash column with 3 x 1 mL water.

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20. Elute GSLs into a new tube with:
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20.1. 2 mL chloroform/methanol (98:2). Push through first 0.5 mL. You can use syringe with adapter that fits into top of column.

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20.2. 2 mL chloroform/methanol (1:3).

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20.3. 1 mL methanol.

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21. Vortex and leave  10 minat 4 °C or carry on to enzymatic digestion.

SECTION: GSL digestion with EGC’ase I
22. Vortex (5 mL, C18) and dry down samples under a stream of nitrogen in heating block (42 °C).

SECTION: GSL digestion with EGC’ase I
23. When about 150 µL sample remaining, transfer to 1.5 mL screw-cap tube.

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24. Rinse sample tube with 200 µL C:M 2:1, vortex and combine with the rest of the sample in the screw-cap tube.

SECTION: GSL digestion with EGC’ase I
26. Dry down, under a slow stream of nitrogen in heating block (42 °C).

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27. Re-suspend in 50 µL C:M 2:1, vortex, dry down under a very slow stream of nitrogen.
 


SECTION: GSL digestion with EGC’ase I
28.  Add 90 µL enzyme/buffer to each sample and vortex:

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28.1. rEGCase : 5 µL enzyme (stored in freezer) plus 85 µL buffer per sample.

 

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29. Incubate at 37 °C for 960 min.
 

SECTION: 2AA labelling of glycans released from GSLs
30. 
 


 

SECTION: 2AA labelling of glycans released from GSLs
31. Add 310 µL labelling mix to the 90 µL sample (digest) in 1.5 mL screw-cap tube.

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32. Incubate in oven at 80 °C for 60 min, vortexing at 30 min.

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33. Allow to cool to 80 Room temperature.

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34. Add 1 mL acetonitrile:water (97:3).

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35. Transfer from screw-cap tube to 15 mL tubes.

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36. Rinse screw-cap tube with 2 x 1 mL acetonitrile:water (97:3) and add to 15 mLtube.

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37. 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

SECTION: 2AA labelling of glycans released from GSLs
38. Apply samples (3.4 mL) to equilibrated DPA-6S columns, let drip through gravity flow.

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39. 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.

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40. Elute with 600 µL water into new tubes.

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41.  Add 60 µL:140 µL (sample:acetonitrile) to HPLC vials and vortex.  

SECTION: HPLC protocol
44. 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). 

 

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45. 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.

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46. The solid phase used is a 4.6 × 250 mm TSK gel-Amide 80 column maintained at 30 °C (Anachem, Luton, UK). 

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47. The chromatographic flow rate is 0.8 mL/min, and run time was 60 min. The total run time is 60 min. 

 
 
 
 
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48. The mobile phases are acetonitrile (solvent A), de-ionised water (solvent B) and 100 millimolar (mM) ammonium acetate, pH 3.85 (solvent C).

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49. 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. 6 min 

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50. 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 %. 29 min 

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51. 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.  4 min 

SECTION: 2AA labelling of glycans released from GSLs
43. DPA-6S eluate is stable stored at -20 °C.

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42. For HPLC, inject 5 µL-50 µL.
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53. At 42 mins the flow-rate is increased to 1.2 mL/min and maintained for 12 min up to 54 min. 12 min 

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54. Between 54 and 60 min the flow-rate is gradually returned to 0.8 mL/min. 6 min  

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55. Individual GSL species are identified by their glucose unit values (GUs), calculated with the HPLC Empower software using a homopolymer dextran ladder (Fig. 1). 

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52. Between 39 and 41 min the solvent ratios are returned to the starting conditions and then maintained for 1 min.  3 min 

SECTION: Figures 1- 4: HPLC profile and sugar structures for GSLs in animal tissues together with the GSL biosynthetic pathway
56. 


 





 



 

 



 


 


 

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55.1. To help identify GSL peaks in the samples, compare the GUs of the peaks in the samples with those in a GSL standard mix.  

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55.2. 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 µLMatreya 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 µLMatreya GM2 Ganglioside  Cat No 1502  (GM2)                                                     0.5 mg/ml
30 µLSigma Asialo-GM2               Cat No G9398 (GA2)                                                    1.0 mg/ml

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55.3. Dry down the mixture under nitrogen and then digest, label and clean up as done for the samples in steps 25-41.
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55.4. Aliquot and store at -20 °C.

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55.5. 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).
SECTION: GSL preparation from animal tissues
1. Homogenise tissue in ddH2O yielding tissue concentration of 25 mg tissue (wet weight) per 1 mL ddH2O. 

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2. Measure protein concentration using the Bicinchoninic acid assay (BCA) method in triplicate at 2 fold dilution (repeat if triplicate values are not in agreement).
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3. Using determined protein concentrations, prepare sample as 200 µg protein in 200 µL ddH2O in a 1.5 ml screw-cap tube.

SECTION: GSL digestion with EGC’ase I
25. Rinse sample tube with 200 µL chloroform, vortex and combine with the rest of the sample in the screw-cap tube.
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5. Vortex.