TITLE: Analysis of glycosphingolipids from human plasma
AUTHORS: David A Priestman, Danielle te Vruchte, Kerri-Lee Wallom, María E Fernández-Suárez, Maria Leondaraki, Carissa Drake , Frances M Platt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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 plasma 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.</div></div>

[STEPS]
?. [GSL preparation from plasma]
Use  plasma for GSL quantification.
50 µl
?. [GSL preparation from plasma]
Add  de-ionised water to make the volume up to  in a 1.5 ml screw-cap tube.
150 µl
200 µl
?. [GSL preparation from plasma]
Add  of chloroform/methanol (1:2, v/v) to give (C/M/W 4:8:3 final).
0.8 mL
Chloroform and methanol are very toxic, and methanol is flammable. Refer to the Safety Data Sheets.
?. [GSL preparation from plasma]
Leave at .
4 °C
?. [GSL preparation from plasma]
Vortex.
?. [GSL preparation from plasma]
Centrifuge at 16,000 x g for  at .
4 Room temperature
?. [GSL preparation from plasma]
Transfer supernatant (about ) to new tube and separate into two phases: by adding  PBS and then  chloroform.
1 mL
0.2 mL
0.2 mL
?. [GSL preparation from plasma]
Vortex.
?. [GSL preparation from plasma]
Centrifuge at 16,000 x g for   at room temperature.
?. [GSL preparation from plasma]
Remove very carefully the lower phase to a new tube and retain the upper phase.
?. [GSL preparation from plasma]
Dry down the lower phase under a stream of (oxygen-free) nitrogen in heating block ().
42 °C
?. [GSL preparation from plasma]
When dry, re-suspend the lower phase in  chloroform/methanol (1:3).
20 µl
?. [GSL preparation from plasma]
Add upper phase to lower phase and vortex.
?. [GSL preparation from plasma]
Pre-equilibrate C18 columns (telos, Kinesis, UK) with 4 x  methanol and 3 x  deionised water.
1 mL
1 mL
?. [GSL preparation from plasma]
Load lower/upper phase mix onto column, let drip through gravity flow.
?. [GSL preparation from plasma]
Rinse sample tube with  water, apply to column to wash.
1 mL
?. [GSL preparation from plasma]
Wash column with 3 x  water.
1 mL
?. [GSL preparation from plasma]
Elute GSLs into a new tube with:
?. [GSL preparation from plasma]
chloroform/methanol (98:2). Push through first . You can use syringe with adapter that fits into top of column.
2 mL
0.5 mL
?. [GSL preparation from plasma]
chloroform/methanol (1:3).
2 mL
?. [GSL preparation from plasma]
methanol.
1 mL
?. [GSL preparation from plasma]
Vortex and leave  at  or carry on to enzymatic digestion.
4 °C
?. [GSL digestion with EGC’ase I]
Vortex (, C18) and dry down samples under a stream of nitrogen in heating block ().
5 mL
42 °C
?. [GSL digestion with EGC’ase I]
When about  sample remaining, transfer to  screw-cap tube.
150 µl
1.5 mL
?. [GSL digestion with EGC’ase I]
Rinse sample tube with  C:M 2:1, vortex and combine with the rest of the sample in the screw-cap tube.
200 µl
?. [GSL digestion with EGC’ase I]
Dry down, under a slow stream of nitrogen in heating block ().
42 °C
?. [GSL digestion with EGC’ase I]
Re-suspend in  C:M 2:1, vortex, dry down under a very slow stream of nitrogen.
50 µl
NB It is essential that ALL the sample is dried in the bottom of the tube.
?. [GSL digestion with EGC’ase I]
Add  enzyme/buffer to each sample and vortex:
90 µl
?. [GSL digestion with EGC’ase I]
rEGCase :  enzyme (stored in freezer) plus  buffer per sample.
4 µl
86 µl
?. [GSL digestion with EGC’ase I]
rEGCase buffer (stored at ): 0.6 % Triton in  sodium acetate pH 5.2.
4 °C
?. [GSL digestion with EGC’ase I]
Incubate at  for .
37 °C
rEGCaseI:  recombinant Endoglycoceramidase I was custom synthesised by GenScript.
?. [2AA labelling of glycans released from GSLs]
Dissolve 2AA in labelling buffer first with vortexing.
Labelling mix: 30 mg/mL 2AA in labelling buffer and 45 mg/mL sodium cyanoborohydrideLabelling buffer: 4% sodium acetate and 2% boric acid in methanol
?. [2AA labelling of glycans released from GSLs]
Add 2AA in buffer to sodium cyanoborohydride and vortex.
Sodium cyanoborohydride is very toxic. Refer to the Safety Data Sheet.
?. [2AA labelling of glycans released from GSLs]
Add  labelling mix to the  sample (digest) in  screw-cap tube.
310 µl
90 µl
1.5 mL
?. [2AA labelling of glycans released from GSLs]
Incubate in oven at  for , vortexing at .
80 °C
?. [2AA labelling of glycans released from GSLs]
Allow to cool to .
80 Room temperature
?. [2AA labelling of glycans released from GSLs]
Add  acetonitrile:water (97:3).
1 mL
?. [2AA labelling of glycans released from GSLs]
Transfer from screw-cap tube to  tubes.
15 mL
?. [2AA labelling of glycans released from GSLs]
Rinse screw-cap tube with 2 x  acetonitrile:water (97:3) and add to tube.
1 mL
15 mL
?. [2AA labelling of glycans released from GSLs]
Pre-equilibrate 50 mg Discovery® DPA-6S SPE Tube (supplied by Sigma-Aldrich) with: acetonitrile2 x  water2 x  acetonitrile
1 mL
1 mL
1 mL
?. [2AA labelling of glycans released from GSLs]
Apply samples () to equilibrated DPA-6S columns, let drip through gravity flow.
3.4 mL
?. [2AA labelling of glycans released from GSLs]
Wash columns with acetonitrile:water (95:5) → add  into the  tubes to wash and add to columns, and then add 3 x  acetonitrile:water (95:5) directly onto the columns.
1 mL
15 mL
1 mL
?. [2AA labelling of glycans released from GSLs]
Elute with  water into new tubes.
600 µl
?. [2AA labelling of glycans released from GSLs]
Add : (sample:acetonitrile) to HPLC vials and vortex.
60 µl
140 µl
?. [HPLC protocol]
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).
Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labelingDCA Neville, V Coquard, DA Priestman, DJM te Vruchte, DJ Sillence, Raymond A Dwek, Frances M Platt, Terry D ButtersAnalytical biochemistry 331 (2), 275-282, (2004). doi: 10.1016/j.ab.2004.03.051.
?. [HPLC protocol]
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.
?. [HPLC protocol]
The solid phase used is a 4.6 × 250 mm TSK gel-Amide 80 column maintained at  (Anachem, Luton, UK).
30 °C
?. [HPLC protocol]
The chromatographic flow rate is 0.8 mL/min, and run time was 60 min. The total run time is .
?. [HPLC protocol]
The mobile phases are acetonitrile (solvent A), de-ionised water (solvent B) and  ammonium acetate,  (solvent C).
?. [HPLC protocol]
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.
?. [HPLC protocol]
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 %.
?. [HPLC protocol]
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.
?. [2AA labelling of glycans released from GSLs]
DPA-6S eluate is stable stored at .
-20 °C
?. [2AA labelling of glycans released from GSLs]
For HPLC, inject -.
5 µl
50 µl
?. [HPLC protocol]
At 42 mins the flow-rate is increased to 1.2 mL/min and maintained for 12 min up to 54 min.
?. [HPLC protocol]
Between 54 and 60 min the flow-rate is gradually returned to 0.8 mL/min.
?. [HPLC protocol]
Individual GSL species are identified by their glucose unit values (GUs), calculated with the HPLC Empower software using a homopolymer dextran ladder (Fig. 2).
?. [HPLC protocol]
Between 39 and 41 min the solvent ratios are returned to the starting conditions and then maintained for 1 min.
?. [Figures 1- 4:  HPLC profile and sugar structures for GSLs in human plasma together with the GSL biosynthetic pathway]
Figures created with BioRender.com
?. [HPLC protocol]
To help identify GSL peaks in the plasma samples, compare the GUs of the peaks in the plasma samples with those in a GSL standard mix.
?. [HPLC protocol]
Prepare a mixture of commercially available authentic GSLs in a screw-cap tube: Matreya Neutral GSL mix    Cat No 1505  (Glc-Cer, Lac-Cer, Gb3, Gb4)              1.0 mg/mlMatreya Ganglioside mix  Cat No 1510  (Lac-Cer, GM3, GD3)                            0.5 mg/ml Matreya Ganglioside mix  Cat No 1511  (GA1, GM1a, GD1a, GD1b,GT1b)      0.5 mg/mlMatreya GM2 Ganglioside  Cat No 1502  (GM2)                                                     0.5 mg/mlSigma Asialo-GM2               Cat No G9398 (GA2)                                                    1.0 mg/ml
50 µl
100 µl
100 µl
30 µl
30 µl
?. [HPLC protocol]
Dry down the mixture under nitrogen and then digest, label and clean up as done for the plasma samples in steps 25-41.
?. [HPLC protocol]
Aliquot and store at.
-20 °C
?. [HPLC protocol]
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).