Protocol 1A Homogenisation of organs

For screening the glycans in mammalian tissues, 100mg - 400mg of tissue is sufficient
for a series of MS analyses including sequential exoglycosidase digestions, MS/MS
studies and linkage analyses. As a rough guide high quality mapping data can be
obtained from 10% of a single mouse kidney.

A compact homogeniser (X120 stand mounted 125 watt; electronic speed between
10,000 and 30,000rpm) with a dispersion shaft is suitable for all tissues.

Cleaning the homogeniser: Prepare ~25ml of an aqueous 80% methanol solution in a
50ml Falcon® (Blue MaxTM) polystyrene tube. Immerse the tip of the homogeniser into
the solution and blast for ~ 60s. Remove homogeniser from power supply and examine
the tip of the dispersion shaft for any residual debris. If any debris is present carefully
remove with a needle. Once removed immerse the tip into the solution and blast for a
further ~ 30s. Repeat this process if required.

Prepare a 0.1M Tris buffer (~0.61g /50ml), pH 7.4 (adjusted with dilute acetic acid,).
Remove 2.5ml of the solution and add 2.5ml of a 10% SDS or CTAB solution to make
a 0.5% w/v detergent solution.

Place organ/tissue into a 50ml Falcon® tube, add ~10ml of the detergent solution and
put the tube in ice. Immerse the tip of the homogeniser to the bottom of the Falcon®
tube and homogenise for ~2 mins to ensure tissue is homogenised. Seal the Falcon®
tube, put on ice and take it to the next stage of the protocol (Protocol 1C).
Protocol 1B Sonication of cells
 Make up an extraction buffer consisting of 1% CHAPS ( 0.5g), 5mM EDTA (0.09g)
and 25mM Tris (0.15g) and adjust to pH 7.4 using dilute acetic acid.
 Approximately 2ml of extraction buffer is added to the cell pellets in 15 ml Falcon®
tubes. The tubes are then placed on ice in preparation for sonication.
 Clean the sonicator head sequentially in 80% aqueous methanol, Milli-Q water and
CHAPS extraction buffer.
 Immerse the tip of the sonicator in the cell/detergent mixture and sonicate for 10
seconds (continuous mode, 40 Amps). Repeat this process five times. Seal the Falcon®
tube, put on ice and take it to the next stage of the protocol (Protocol 1C).
Protocol 1C Dialysis

Rinse out a large glass beaker and a magnetic flea with Milli-Q water. Prepare 3.5
litres of 50mM buffer by dissolving 13.8g of ammonium hydrogen carbonate in 3.5
litres of Milli-Q water.

Transfer the homogenate / cell lysate into high quality regenerated cellulose dialysis
tubing (molecular weight cut off point of 12-14kDa), pre-soaked in Milli-Q water.

Dialyse on a magnetic stirrer in a 4oC cold room with regular changes of buffer (2-3
times) over a period of 48h. Transfer contents to glass culture tubes and lyophilize.
PROTOCOL SET 2 PREPARATION OF GLYCOPEPTIDES
Protocol 2A Reduction and Carboxymethylation

Prepare a 0.6M Tris buffer (~3.63g/50 ml), pH 8.5 (adjusted with acetic acid) and
bubble nitrogen through for 30min.

Incubate the lyophilized sample for 45min at 37oC in 1.5ml of 2mg/ml dithiothreitol
(DTT) in degassed Tris buffer (0.6M, pH 8.5).

Add 1.5ml of 12mg/ml iodoacetic acid (IAA) and leave the reduced sample to stand in
the dark at room temperature for 90min.

Terminate the reaction by dialysing against ammonium hydrogen carbonate buffer
(50mM, 4.5 litres) at 4oC for 16-24h (see protocol 1C). Lyophilize.
Protocol 2B Tryptic digestion



Prepare 50mM ammonium hydrogen carbonate buffer at pH 8.4 (adjust pH with
diluted ammonia solution) fresh for each analysis.
Weigh out 0.5-1.5mg of TPCK treated bovine pancreas L-1-tosylamide-2phenylethylchloromethyl ketone trypsin and add the appropriate amount of ammonium
hydrogen carbonate (50mM, pH 8.4) buffer to make a 2mg in 1.5mL solution of
trypsin. Add sufficient quantities of this solution (1500l) to the carboxymethylated
lyophilized sample so that the sample is completely suspended in the solution.
Incubate at 37oC for 4-8h. Typically, a 1:50/1:100 ratio of enzyme to protein (w/w) is
required to digest the sample.
The resulting mixture is purified by Sep-Pak® C18 (Waters Corporation) using the
propan-1-ol / 5% acetic acid system (Protocol 2C). Do not lyophilize sample
homogenates after digest, instead add a few drops of 5% acetic acid and load the
sample directly onto a conditioned Sep-Pak® C18 (Waters Corporation) as the
lyophilized samples can be difficult to dissolve.
Protocol 2C Sep-Pak® C18 purification: Propan-1-ol / 5% acetic acid system

Prepare the following solutions: 5% acetic acid, 20% propan-1-ol in 5% acetic acid,
40% propan-1-ol in 5% acetic acid

Condition the Sep-Pak® C18 (Waters Corporation) cartridge by eluting successively
with methanol (5ml), 5% acetic acid (5ml), propan-1-ol (5ml) and 5% acetic acid (3 x
5ml). * Sep-Pak® C18 cartridges are attached to 10 ml syringes.

Load the sample directly onto to the Sep-Pak.

Wash with ~ 30ml of 5% acetic acid. Elute stepwise with 3-4ml each of 20% propan1-ol in 5% acetic acid, 40% propan-1-ol in 5% acetic acid and 100% propan-1-ol.

Collect the fractions in culture tubes. Cover with pre-pierced Parafilm® and evaporate
to dryness.
PROTOCOL SET 3 RELEASE OF GLYCANS
PROTOCOL 3A PNGase F digestion

Combine the 20% propan-1-ol in 5% acetic acid, 40% propan-1-ol in 5% acetic acid
fractions from protocol 2C in 200l of ammonium hydrogen carbonate solution
(50mM, pH 8.4).

Add PNGase F (3U of PNGase F), and incubate at 370C for 20h.

Freeze-dry the PNGase F digestion.

Purify the digest using protocol 2C as follows: omit the washing step and elute with
5ml of 5% acetic acid followed by 4ml of 20% propan-1-ol in 5% acetic acid. Dry
down the 5% acetic acid fraction (the N-glycan fraction) and the 20% fraction
(containing the peptides/O-linked glycopeptides).
PROTOCOL 3B Reductive elimination

Prepare a 0.05M aqueous NaOH stock solution by dissolving 0.2g of NaOH in 100ml
of Milli-Q water.

Prepare a batch of pre-washed Dowex (50W-X8 (H) 50-100 mesh) beads. Place 100g
of beads in a 250ml glass bottle. Add ~ 100 ml of 4M HCl and decant. Repeat this two
more times then wash beads by adding, agitating and decanting the beads ~ 25-30
times with Milli-Q water to remove residual HCl. Wash the beads 3 times with 5%
acetic acid (150ml each) and leave the beads immersed in~ 200ml of 5% acetic acid.
The treated beads can be kept equilibrated in this state for many months.

Prepare a 1.0M sodium borohydride solution by dissolving sodium borohydride in
0.05M NaOH solution to a concentration of 38-40 mg/ml.

Add 400l of the sodium borohydride solution to the sample and incubate at 45oC in a
teflon lined screw capped culture tube overnight for 16 h.

Terminate the reaction by adding glacial acetic acid dropwise until the fizzing stops
(usually 5 drops).

Desalt the O-glycans from the mixture as follows:

Clamp the desalting column (a Pasteur pipette plugged at the tapered end with a small
amount of glass wool) in a retort stand. Insert the tapered end of the Pasteur pipette in
a piece of silicone tubing (~10cm length with internal bore diameter of 1-2mm) and
clip the end of the small piece of tubing with an adjustable clip so that the tube is
blocked.

Place 5% acetic acid in the column and open the clip slightly to let the acid slowly
flow out. As the acid runs out, fill the column with Dowex beads. Fill to the
constriction point of the Pasteur.

Slowly pass approximately 20 ml of 5% acetic acid over the Dowex to wash it. Close
the clip and load the sample onto the top of the column.

Place a culture tube under the column and open the clip to allow the sample to slowly
run onto the column.

Just as the sample has fully passed onto the column close the clip and load the top of
the column with 5% acetic acid.

Open the clip again and allow the liquid to flow through the column and collect in the
tube. As the acetic acid level drops, top up and collect approximately 5ml of eluent.
Freeze-Dry.

Excess borates are removed from the O-glycans by co-evaporating the borates with a
10% acetic acid in methanol solution (3 x 0.5ml) under a stream of nitrogen at room
temperature. Repeat twice more. After the third drying step the sample is ready for
derivatization.
PROTOCOL SET 4 PERMETHYLATION
* First prepare an anhydrous dimethyl sulphoxide (DMSO) stock solution. Stand DMSO
over calcium hydride overnight or longer in a 500ml round bottom Quick-fit flask until the
calcium hydride has settled to the bottom of the flask. Store the DMSO over fresh calcium
hydride at room temperature. Keep the stopper tight and replace immediately each time
after the DMSO has been dispensed.
Protocol 4A Sodium Hydroxide Permethylation

Make sure the sample to be permethylated is completely dry and the cap of the screwcapped culture tube is teflon-lined.

Place 5 pellets of NaOH in a dry mortar and add approximately 3 ml of anhydrous
DMSO using a Pasteur pipette.

Use a pestle to grind the NaOH pellets until a slurry is formed with the DMSO.

Take up 0.5-1ml of the DMSO/NaOH slurry with a Pasteur pipette and add to the
sample.

Add about 0.2-0.5ml of methyl iodide (or deuteromethyl iodide).

Mix vigorously and then agitate the reaction mixture on an automatic shaker for 10
min. at room temperature.

Quench the reaction by slow dropwise additions of about 1ml of water with constant
shaking between additions to lessen the effects of the highly exothermic reaction.

Add 1ml of chloroform and make up the total volume to ~3ml with water. Mix
thoroughly and allow the mixture to settle into two layers. Centrifuge the mixture to
assist better separation, if necessary.

Remove and discard the upper aqueous layer.

Wash the lower chloroform layer several times with water until the water being
removed is completely clear.

Dry down the chloroform layer under a gentle stream of nitrogen. Purify the dried
mixture by Sep-Pak® C18 using the aqueous acetonitrile system (Protocol 4B).
Protocol 4B Sep-Pak® C18 purification: Aqueous acetonitrile system

Prepare the following solutions: 15% acetonitrile in Milli-Q water, 35% acetonitrile in
Milli-Q water, 50% acetonitrile in Milli-Q water, 75% acetonitrile in Milli-Q water.

Condition the Sep-Pak® cartridge by eluting successively with methanol (5 ml), MilliQ water (5ml), acetonitrile (5ml) and water (3 x 5ml).

Re-dissolve the sample in 1:1 methanol:water (200µl) and load onto the Sep-Pak®
cartridge.

Elute stepwise with 5ml of water and 2ml each of 15%, 35%, 50% and 75% aqueous
acetonitrile. Collect each fraction in culture tubes, cover with pre-pierced Parafilm®
and evaporate to dryness.

The samples are now ready for MS analysis. Permethylated glycans are usually present
in the 35%, 50% and 75% acetonitrile fractions.
PROTOCOL SET 5: Mass spectrometry analysis
Protocol 5A: Matrix assisted laser desorption ionisation (MALDI)
MALDI MS is performed in positive reflectron mode using a ABI Voyager-DE STR
MALDI-TOF. Derivatised carbohydrate samples are dissolved in ~75l methanol/water
8:2 (v/v) and mixed in a 1:1 ratio with ~10mg/ml 2,5-dihydroxybenzoic acid in 80:20 (v/v)
methanol/water. 1.5l aliquots are spotted onto a 100-well sample plate and dried with a
heat gun. Angiotensin I, adrenocorticotropic hormone fragment (ACTH) 1-17, ACTH
fragment 18-39, ACTH Fragment 7-38, are used for external calibration.
MALDI parameters
Mass range: m/z 500-5000
Low mass gate: 250
Accelerating volts: 20000
Grid: 77.5%
Guide wire: 0.002%
Delay time: 250 nsec
Protocol 5B: Electrospray ionisation QTOF MS and MS/MS analysis
MS and MS/MS data are acquired on a quadrupole orthogonal acceleration time of flight
(Q-TOF) mass spectrometer (Micromass, UK) fitted with a Z-spray API source and
operated in positive ion mode. 5l of permethylated glycans in methanol are loaded into
NanoES spray capillaries (Proxeon, Denmark). A potential of 1.5kV is applied to the
nanoflow tip to produce a flow rate of between 10-30 nanolitres/minute. Drying and
collision gases are nitrogen and argon respectively, and the collision gas pressure is
maintained at 10-4mbar. Collision energies vary between 30eV and 90eV, depending on
the m/z value of the ion selected for CAD. The data are acquired and processed using
Masslynx software (Micromass, UK). The Q-TOF is calibrated using a 1pmol/µl solution
of [Glu1]-fibrinopeptide B in acetonitrile / 5% acetic acid (1:3 [v/v]).