TITLE: Analysis of glycosphingolipids from human cerebrospinal fluid
AUTHORS: David A Priestman, Reuben Bush, Maria Leondaraki, 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 control and patient cerebrospinal fluid 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.

[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 cerebrospinal fluid
1. Use 100 µL cerebrospinal fluid for GSL quantification.

SECTION: GSL preparation from cerebrospinal fluid
2. Add 100 µL de-ionised water to make the volume up to 200 µL in a 1.5 ml screw-cap tube.

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

 

SECTION: GSL preparation from cerebrospinal fluid
4. Leave at 4 °C for 960 min 
SECTION: GSL preparation from cerebrospinal fluid
5. Vortex.
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6. Separate into two phases: by adding 0.2 mL PBS and then 0.2 mL chloroform.

SECTION: GSL preparation from cerebrospinal fluid
7. Vortex.
SECTION: GSL preparation from cerebrospinal fluid
8. Centrifuge at 16,000 x g for 10 min  at room temperature.

SECTION: GSL preparation from cerebrospinal fluid
9. Remove very carefully the lower phase to a new tube and retain the upper phase.

SECTION: GSL preparation from cerebrospinal fluid
10. Dry down the lower phase under a stream of (oxygen-free) nitrogen in heating block (42 °C).

SECTION: GSL preparation from cerebrospinal fluid
11. When dry, re-suspend the lower phase in 20 µL chloroform/methanol (1:3).

SECTION: GSL preparation from cerebrospinal fluid
12. Add upper phase to lower phase and vortex.
SECTION: GSL preparation from cerebrospinal fluid
13. Pre-equilibrate C18 columns (telos, Kinesis, UK) with 4 x 1 mL methanol and 3 x 1 mL deionised water.

SECTION: GSL preparation from cerebrospinal fluid
14. Load lower/upper phase mix onto column, let drip through gravity flow.
SECTION: GSL preparation from cerebrospinal fluid
15. Rinse sample tube with 1 mL water, apply to column to wash.

SECTION: GSL preparation from cerebrospinal fluid
16. Wash column with 3 x 1 mL water.

SECTION: GSL preparation from cerebrospinal fluid
17. Elute GSLs into a new tube with:
SECTION: GSL preparation from cerebrospinal fluid
17.1. 2 mL chloroform/methanol (98:2). Push through first 0.5 mL. You can use syringe with adapter that fits into top of column.

SECTION: GSL preparation from cerebrospinal fluid
17.2. 2 mL chloroform/methanol (1:3).

SECTION: GSL preparation from cerebrospinal fluid
17.3. 1 mL methanol.

SECTION: GSL preparation from cerebrospinal fluid
18. Vortex and leave  10 minat 4 °C or carry on to enzymatic digestion.

SECTION: GSL digestion with EGC’ase I
19. 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
20. When about 150 µL sample remaining, transfer to 1.5 mL screw-cap tube.

SECTION: GSL digestion with EGC’ase I
21. 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
23. Dry down, under a slow stream of nitrogen in heating block (42 °C).

SECTION: GSL digestion with EGC’ase I
25. 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
26.  Add 90 µL enzyme/buffer to each sample and vortex:

SECTION: GSL digestion with EGC’ase I
26.1. rEGCase : 5 µL enzyme (stored in freezer) plus 85 µL buffer per sample.

SECTION: GSL digestion with EGC’ase I
26.2. rEGCase buffer (stored at 4 °C): 0.6 % Triton in 50 millimolar (mM) sodium acetate pH 5.2.

SECTION: GSL digestion with EGC’ase I
27. Incubate at 37 °C for 960 min.
 

SECTION: 2AA labelling of glycans released from GSLs
28. 
 

Dissolve 2AA in labelling buffer first with vortexing.
SECTION: 2AA labelling of glycans released from GSLs
29. Add 2AA in buffer to sodium cyanoborohydride and vortex.

 

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

SECTION: 2AA labelling of glycans released from GSLs
31. Incubate in oven at 80 °C for 60 min, vortexing at 30 min.

SECTION: 2AA labelling of glycans released from GSLs
32. Allow to cool to 80 Room temperature.

SECTION: 2AA labelling of glycans released from GSLs
33. Add 1 mL acetonitrile:water (97:3).

SECTION: 2AA labelling of glycans released from GSLs
34. Transfer from screw-cap tube to 15 mL tubes.

SECTION: 2AA labelling of glycans released from GSLs
35. Rinse screw-cap tube with 2 x 1 mL acetonitrile:water (97:3) and add to 15 mLtube.

SECTION: 2AA labelling of glycans released from GSLs
36. 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
37. Apply samples (3.4 mL) to equilibrated DPA-6S columns, let drip through gravity flow.

SECTION: 2AA labelling of glycans released from GSLs
38. 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.

SECTION: 2AA labelling of glycans released from GSLs
39. Elute with 600 µL water into new tubes.

SECTION: 2AA labelling of glycans released from GSLs
41.  Resuspend in 60 µL  of 70:30 acetonitrile:water

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). 

 

SECTION: HPLC protocol
43. 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.

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

SECTION: HPLC protocol
46. The chromatographic flow rate is 0.8 mL/min, and run time was 60 min. The total run time is 60 min. 

   
 
 
SECTION: HPLC protocol
47. 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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48. 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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49. 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 

SECTION: HPLC protocol
50. 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
42. For HPLC, inject 50 µL.
SECTION: HPLC protocol
52. 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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53. Between 54 and 60 min the flow-rate is gradually returned to 0.8 mL/min. 6 min  

SECTION: HPLC protocol
54. Individual GSL species are identified by their glucose unit values (GUs), calculated with the HPLC Empower software using a homopolymer dextran ladder (Fig. 2). 

SECTION: HPLC protocol
51. 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- 5:  HPLC profile and sugar structures for GSLs in cerebrospinal fluid together with the GSL biosynthetic pathway
55. 







  




 
 
 
 
  

 

SECTION: HPLC protocol
54.1. To help identify GSL peaks in the CSF samples, compare the GUs of the peaks in the CSF samples with those in a GSL standard mix.  

SECTION: HPLC protocol
54.2. 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

SECTION: HPLC protocol
54.3. Dry down the mixture under nitrogen and then digest, label and clean up as done for the CSF samples in steps 25-41.
SECTION: HPLC protocol
54.4. Aliquot and store at-20 °C.

SECTION: HPLC protocol
54.5. 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: 2AA labelling of glycans released from GSLs
40. Dry down under a slow stream of nitrogen in a heating block (42 °C ).

SECTION: GSL digestion with EGC’ase I
22. Rinse sample tube with 200 µL chloroform, vortex and combine with the rest of the sample in the screw-cap tube.
SECTION: GSL digestion with EGC’ase I
24. Re-suspend in 200 µL C:M 2:1, vortex, dry down under a very slow stream of nitrogen.