Glycoprotein methods protocols - biotechnology
Trang 115
O-Linked Oligosaccharide Chain Release
and Fractionation
Elizabeth F Hounsell
1 Introduction
O-linked chains of glycoproteins have classically been released by alkaline
boro-hydride degradation, in which mild alkali (0.05 M OH–) is used to cause β-elimination from the β carbon of serine (R-H) or threonine (R-CH3) in the protein backbone.
To inhibit subsequent elimination around the glycosidic ring of the linkage monosac-charide, and on backward down the oligosaccharide if linked at C-3, the reaction is
carried out in the presence of 1 M sodium borohydride to give simultaneous reduction
of the reducing sugar formed after elimination An advantage of this technique is that there is now a large database of nuclear magnetic resonance (NMR) chemical shifts
for mucin-type oligosaccharide alditols (1,2) that can be searched in a computer-as-sisted way (3) for structural identification The disadvantage of this technique is that
the resulting alditol is not capable of undergoing a reductive amination procedure for coupling to a sensitive flourescent label or for polyvalent coupling to lipid or protein for subsequent immunoassay However, it is possible to reoxidize selectively the alditol using periodate, which results in a new aldehyde being formed for reductive amination, and, indeed, this is the basis for a method for structural analysis by
thin-layer chromatography-mass spectometry (MS) (4) Note, however, that for subsequent
biological or immunological assay, any branching at GalNAc-Ser/Thr, often found in mucins, is destroyed by this procedure.
From: Methods in Molecular Biology, Vol 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A Corfield © Humana Press Inc., Totowa, NJ
Trang 2If branching is not present, a useful cleavage can be obtained by the enzyme
O-glycanase ( α-N-acetylgalactosaminidase) This enzyme has the disadvantage that only
relatively simple oligosaccharides are released with any reproducibility (5,6); the
dis-accharide Gal β1-3GalNAcα1-Ser/Thr is the best substrate Hydrazinolysis is prob-ably the preferred technique for release of all oligosaccharides in both the presence and the absence of sulfate and/or sialic acids, if the conditions are optimized It may be
a good idea to carry out this reaction in triplicate with the alkaline borohydride
degra-dation and O-glycanase in order to ensure that the complete picture of a mucin at a
structural level is obtained Note that if the protein is required for analysis, rather than
the oligosaccharide, mild alkali in the absence of borohydride (7) or hydrolysis with
trifluoromethanesulfonic acid (TFMSA) can be used to keep the protein intact After the release of oligosaccharide alditols, these can be fractionated by normal- or
re-versed-phase high-performance liquid chromatography (HPLC) (8) or anion-exchange
chro-matography, e.g., high-performance anion-exchange chromatography (HPAEC) on two
CarboPac PA-100 columns (Dionex Camberley, Surrey, UK) in series (9) Sulfated and
sialylated oligosaccharide alditols can also be separated by these techniques; however, for better results the reversed-phase HPLC should be by porous graphitized carbon (PGC) in
0.1% trifluoroacetic acid (TFA) (10,11), and the normal-phase HPLC should be in the pres-ence of buffers (9) Sulfated oligosaccharide alditols are retained well on the HPLC columns
(12) For reducing oligosaccharides, PGC is again a good alternative for both neutral and
anionic oligosaccharides, which have similar retention times to each other and to their alditols.
On HPAEC (13), the reducing oligosaccharides will be retained significantly longer than the
alditols, to give improved chromatography Sulfated oligosaccharides may now be retained too long on a CarboPac PA-100 column Although HPAEC with pulsed amperometric detec-tion (PAD) is a sensitive technique, addidetec-tional sensitivity can be obtained by flourescent
labeling The usual labels, 2-aminopyridine (14,15) and 2-aminobenzamide (15,16) cause
sulfated oligosaccharides to be retained too long on reversed phase or PGC column For neutral or sialylated oligosaccharides, separation is primarily achieved by reversed-phase
and normal-phase chromatography, often as a two-dimentional map (14,15), or on weak ion exchange, e.g., GlycoSep C™ (Oxford GlycoSciences, Abingdon, Oxford, UK) (17)
Neu-tral 2-aminobenzamide (2-AB) oligosaccharides (naturally occuring or after desialylation) can be analyzed by gel filtration in water as eluent (usually Bio-Gel P4 chromatography) to give molecular size estimation This is particularly useful to follow exoglycosidase diges-tions to obtain additional sequence information More information about these techniques
can be obtained from refs 18–20 The use of electrospray ionization MS coupled with
colli-sion-induced dissociation MS is another possibility for analysis at the glycopeptide level
(21) The internet provides a source for predicting potential O-glycosylation sites on proteins
at http://www.cbs.dtu.dk/netOglyc/cbsnetOglyc.html.
2 Materials
2.1 Alkaline Borohydride Degradation ( β -Elimination)
and Chromatography
1 1 M NaBH4 in 0.05 M NaOH made up fresh.
2 Glacial acetic acid
Trang 33 Methanol.
4 Cation-exchange column Dowex 50W X8 H+ form
5 Phenyl boronic acid (PBA) Bond Elut columns (Jones Chromatography, Hengoed, UK)
activated with MeOH (22).
6 0.2 M NH4OH
7 0.01, 0.1, and 0.5 M HCl.
8 HPLC apparatus fitted with an ultraviolet detector (approx 1 nmol of mono- and
oligosac-charides containing N-acetyl groups can be detected at 195–210 nm) and pulsed
electro-chemical detector (oligo- and monosaccharides ionized at high pH can be detected at picomole level)
9 Columns: reversed-phase C18, amino bonded silica, PGC (Hypercarb 7µ, Hypersil Ltd., Runcorn Cheshire, UK), CarboPac PA100 and CarboPac PA1 (Dionex Camberley)
10 Eluents for HPLC: HPLC grade water, acetonitrile, 0.1% aqueous TFA; acetonitrile con-taining 0.1% TFA, ammonium formate
11 Eluents for HPAEC: 12.5 M NaOH (BDH, Poole, Dorset, UK) diluted fresh each day to
200, 100, 80, and 1.5 mM After chromatography and detection, salt needs to be removed
by a Dionex micromembrane suppressor or by cation-exchange chromatography before further analysis, e.g., by methylation
12 Eluent A: 0.08 M NaOH.
13 Eluent B: 0.5 M sodium acetate (Aldrich, Poole, Dorset, UK) in 0.08 M NaOH.
2.2 Reoxidation
1 Sodium periodate, analytical reagent grade
2 Imidazole (Sigma, Poole, Dorset, UK) 40 mM adjusted to pH6.5 with HCl
3 Butan-2,3-diol
2.3 Release of the Core 1 Disaccharide (Gal β 1-3GalNAc α –) from Mucins
1 Bio-Spin®chromatography columns (Bio-Rad, Hercules CA), or home made columns suitable for 1.5-mL microcentrifuge tubes packed with 0.8 mL of Bio-Gel P30 acrylamide gel matrix Store at 4°C in 0.15 M sodium chloride-17.5 mM sodium citrate, pH 7.0,
containing 0.2% w/v sodium azide as preservative
2 O-Glycanase®; Streptococcus (Diplococcus) pneumoniae Endo- α-N-acetylgalacto-saminidase (EC 3.2.1.907) (Oxford GlycoSciences) Store at –20°C for up to 6 mo, but avoid repeated freeze-thawing
3 0.1 M sodium citrate-phosphate, pH 6.0; make up with HPLC-grade water.
4 Dowex 50X-12 H+-form resin
5 Sephadex GM25
2.4 Hydrazinolysis
1 0.5-mL screw-capped V-bottomed Reacti-vials™ (Pierce and Warriner, Chester, UK)
2 Anhydrous hydrazine (Pierce and Warriner)
3 Whatman CF-11 cellulose chromatography medium
4 Reagent A: butanol:ethanol:acetic acid, 4:1:0.5 (v/v/v)
5 Reagent B: butanol:ethanol:water, 4:1:1 (v/v/v)
6 Reagent C: acetic anhydride:methanol, 2:5 (v/v)
7 Reagent D: 0.2 M sodium acetate.
8 Sep-Pak C18 cartridge (Waters, Watford, UK)
9 Alternatively, the Glycorelease N- and O-Glycan recovery kit (Oxford GlycoSciences)
can be used
Trang 42.5 Isolation of the Protein by TFMSA Destruction of Oligosaccharides
1 Anhydrous TFMSA (23).
2 Reagents as specified in Glyco Free™ Deglycosylation kit (K500, Oxford, Glyco-Sciences)
2.6 2-AB Fluorescence Labeling and Size Exclusion
Chromatography (SEC)
1 Signal labeling kit (K404,Oxford GlycoSciences)
2 Dowex AG50 X 12 (H+ form)
3 Dowex AG1 X 8 (acetate form)
4 RAAM 2000 Glycosequencer (Oxford GlycoSciences) or Biogel P4 column (100 × 2 cm)
in a water jacket at 55°C and HPLC pump with refractive index and fluorescence detectors
5 GlycoSepH™ and GlycoSepC™ HPLC column (Oxford GlycoSciences)
3 Methods
3.1 Alkaline Borohydride Degradation ( β -Elimination)
1 Release O-linked chains by treatment with 0.05 M sodium hydroxide in the presence of 1 M
NaBH4 or NaB[3H]4 for 16 h at 50°C
2 Degrade excess NaBH4or NaB[3H]4by the careful addition with the sample on ice of glacial acetic acid (to pH 7.0) or acetone (1 mL/100 mg of NaBH4) followed by repeated evaporation with methanol
3 Desalt on a cation-exchange column and analyze by reversed-phase HPLC (Subheading
3.6.2.) or HPAEC (Subheading 3.7.1.).
4 Or, for microscale identification of the presence of alditols, dissolve the sample in 200 µL
0.2 M NH4OH and add to the top of a PBA minicolumn prewashed with MeOH, water,
and 0.2 M NH4OH
5 Wash the PBA column with 2 × 100 µL 0.2 M of NH4OH and 2 × 100 µL of water
6 Specifically elute the alditols in 1 M acetic acid.
7 Evaporate the sample and reevaporate with 2 × 100 µL of water
3.2 Oxidation of Oligosaccharide Alditols
1 To the dry alditols, add twice the molar ratio of sodium periodate in imidazole buffer (see
Note 1).
2 Oxidize in the dark at 0°C for 5 min
3 Destroy excess oxidant with butan-2,3-diol (two times the molar excess over periodate) for 40 min at 0°C in the dark
3.3 Release of Core 1 Disaccharide (Gal β 1-3GalNAc α –) from Mucin Using Endo- α - N -Acetylgalactosaminidase ( O -Glycanase)
3.3.1 The Removal of Glycerol from O -Glycanase ( see Note 2)
1 Invert a Bio-Spin P30 polyacrylamide BioGel column (0.8 mL column volume) several times and allow the buffer to drain by gravity
2 Wash the column with 300 µL of 0.01 M citrate-phosphate, pH 6.0, place in a collection tube, and centrifuge for 2 min at 1100g Repeat four times.
3 Make up 6 mU O-Glycanase to 100 µL with 0.01 M citrate-phosphate, pH 6.0, and load
the sample carefully and directly to the center of the column, drop wise (see Note 3).
4 Centrifuge for 4 min at 1100g and collect the excluded O-Glycanase.
Trang 55 Pass the excluded O-Glycanase through a second Bio-Spin column to maximize glycerol
removal
3.3.2 Hydrolysis of Gal β 1-3GalNAc α - from mucin using O -Glycanase
1 Reconstitute mucin (100 µg) with 90 µL of 0.1 M citrate-phosphate, pH 6.0, containing
100µg/mL of bovine serum albumin and 0.02% (w/v) sodium azide Mix well
2 Add 0.6 mU of deglycerolyated O-Glycanase (10 µL)
3 Incubate for 18 h at 37°C (see Note 4).
4 Load reactions on Dowex 50X-12H+form resin (3 × 0.5 cm column) and elute with three column vol of HPLC-grade water
5 Collect the effluent and eluent and then pool (2.5-mL volume)
6 Load onto a PD10-Sephadex GM25 column and elute with HPLC-grade water to isolate liberated disaccharide from intact mucin
3.4 O -Linked Oligosaccharide Release by Hydrazinolysis
1 Dry salt-free glycoprotein into a V-bottomed Reacti-vial and remove from the lyophilizer immediately before the reaction is due to commence
2 Using a clean, dry, acid-washed glass pipet, transfer 100 µL of anhydrous hydrazine to the vial and cap immediately Incubate at 60°C for 5 h (see Note 5).
3 Allow the Reacti-vial to cool to room temperature, and transfer the reaction mixture to a 1-mL cellulose (Whatman CF-11) microcolumn washed with reagent A
4 Wash the column with 3 × 1mL reagent B
5 Re-N-acetylate the glycans on the column by adding 1.4 mL of reagent C for 30 min at
room temperature
6 Wash the column with 4 × 1 mL of reagent B followed by 1 mL of methanol (see Note 6).
7 Elute the oligosaccharides with 2 × 1 mL of reagent D
8 Complete the re-N-acetylation with 0.1 mL of acetic anhydride for 30 min at room
tem-perature
9 Wash a Sep-Pak C18cartridge with 2 mL of methanol and 2 mL of H2O
10 Transfer the sample containing O-glycans to the cartridge and collect the eluate Elute the
remaining glycans with 0.5 mL of H2O
3.5 TFMSA Treatment
1 Make up a 3 g/mL solution of TFMSA in anisole and cool in dry ice/ethanol
2 Add 10 times the weight of TFMSA in anisole to the lyophilized material in a teflon screw-capped vial standing on a bed of ice
3 Incubate at 0°C for 6–16 h with occasional vigorous shaking
4 With the vial on ice, add 1 vol of cold anhydrous diethyl ether and then add this mixture
to 1 vol of a frozen slush of aqueous pyridine
5 Warm the solution to room temperature and extract with ether
6 Collect the aqueous phase containing the peptide with partial glycosylation depending on the reaction time
7 Alternatively, follow the instructions in the GlycoFree kit
3.6 HPLC of 2-AB-Labeled Oligosaccharides
3.6.1 Preparative HPLC on a GlycoSep C HPLC Column
1 Wash the column with water for 30 min at a flow rate of 0.4 mL/min
2 Wash the column with acetonitrile for 30 min at a flow rate of 0.4 mL/min
Trang 63 Wash the column with 0.5 M ammonium acetate, pH 4.5, for 30 min at a flow rate of
0.4 mL/min (see Note 7).
4 Rewash the column with water for 30 min at a flow rate of 0.4 mL/min
5 Equilibrate the column in 75% aqueous acetonitrile at a flow rate of 0.4 mL/min
6 Inject the 2-AB-labeled sample in 75% aqueous acetonitrile with fluorescence detection using an excitation λ = 330 nm and an emission λ = 420 nm
7 Elute the sample with the following gradient with fraction collection at a flow rate of
0.4 mL/min (see Note 8):
a 75% acetonitrile for 5 min
b 62.5% acetonitrile over the next 25 min
c 60% over the next 30 min
d Back to 75% at 60 min for the total run
3.6.2 Porous Graphitized Carbon Chromatography
1 Wash the column with water for 30 min at a flow rate of 0.75 mL/min (see Note 9).
2 Wash the column with acetonitrile for 30 min at a flow rate of 0.75 mL/min
3 Equilibrate the column in 0.2% TFA in 20% aqueous acetonitrile at a flow rate of
0.75 mL/min (see Note 10).
4 Inject the 2-AB-labeled sample in water, with fluorescence detection using an excitation
λ = 330 nm and an emission of λ = 420 nm (see Note 11).
5 Elute the sample with the following gradient at a flow rate of 0.75 mL/min with water/ acetonitrile containing 0.2% TFA:
a 20% acetonitrile for 2 min
b 40% acetonitrile over 33 min
c 30% acetonitrile over another 35 min
d Back to 20% acetonitrile over the next 4 min
6 Store the column in 75:25 (v/v) acetonitrile:water
3.7 HPAEC of Released Oligosaccharides
3.7.1 HPAEC Analysis of Alkaline-Borohydride–Released Mucin
Oligosaccharide Alditols
1 Inject an aliquot (50 and 100 µL) of released mucin oligosaccharide alditols, containing
320 ng of D-melibiose as internal standard, onto the two CarboPac PA-100 columns (con-nected in series) equilibrated in eluent A
2 Elute with an increasing gradient of eluent B at a flow rate of 0.7 mL/min
3 Profile the chromatogram obtained using a range of well-characterized human oligosac-charide alditol standards
4 Reequilibrate the columns before each subsequent sample application
3.7.2 HPAEC Analysis of O -Glycanase-Released Gal β 1-3GalNA c
1 Inject a 1-µg aliquot of the O-Glycanase-released product, containing 320 ng of
D-meli-biose as internal standard, onto the two CarboPac PA100 columns (connected in series) equilibrated in eluent A
2 Elute with an increasing gradient of eluent B at a flow rate of 0.7 mL/min
3 Quantify the Galβ1-3GalNAc released using a calibration curve for standard amounts of Galβ1-3GalNAc (see Note 12).
4 Reequilibrate the column before each subsequent sample application (see Note 13).
Trang 73.8 Fluorescence Labeling with 2-AB
1 Dry salt free glycans into a 0.5-mL Eppendorf tube
2 Prepare labelling reagent of 70% DMSO 30% glacial acetic acid containing 0.25 M of
2-AB and 0.1 M of NaCNBH3(see Note 14).
3 Add 5 µL of labeling reagent and incubate the sample at 65°C for 2 h
4 Centrifuge sample briefly
5 Transfer to a hydrophillic separation disk (supplied with labeling kit) washed with 1 mL
of water, lml 30% acetic acid, and 1 mL of acetonitrile
6 Load the sample onto disk and leave for 15 min (see Note 15).
7 Wash the tube with 100 µL acetonitrile and add to disk
8 Wash disk with 1 mL of acetonitrile followed by 5 × 1 mL 4% water in acetonitrile
9 Elute the sample with 3 × 0.5mL of water (see Note 15).
10 Dry the sample to about 100 µL
11 Prepare 150 µL of AG50-X12 resin in a microcolumn and wash with 5 mL 1.5% triethy-lamine in water followed by 3 × 1 mL water (see Note 16)
12 Add 150 µL of AG1 X8 (acetate form) to the microcolumn taking care not to disturb the AG50 resin
13 Wash with 0.5 mL of water
14 Load the sample in 100 µL water and elute with 4 × 0.4 mL of water
15 Filter the sample through a 0.45-µm filter and dry for further analysis
4 Notes
1 Standard O-linked chains, i.e., those having the linkage
are cleaved by periodate at the C-4—C-5 bond, thus giving a characteristic product for chains linked at the C-3 and/or C-6
2 The efficient removal of glycerol (added as an enzyme stabilizer) from commercial
O-Gly-canase is necessary for the subsequent HPAEC analysis of hydrolyzed Galβ1-3GalNAcα-,
because glycerol was found to be highly PAD active (20) Deglycerolated O-Glycanase,
how-ever, does not store well, so it is best to remove the glycerol on the day of use
3 The optimum volume of sample is between 50 and 100 µL Applying more or less results
in poor recovery of desalted sample Likewise, sample directed down the side of the gel also results in poor recovery
4 1 mU of O-glycanase releases approx 500 ng of Gaβ1-3GalNAc from 1 µg of antifreeze glycopeptides and 50 ng from 1 µg of asialofetuin (20).
5 The reaction should be incubated in either an oven or a heating block but not in a water bath Because of the highly toxic and flammable nature of hydrazine, all manipulations that involve its use should be carried out in a fume cupboard with additional skin and eye protection The evaporator used should also be vented into a fume cupboard; if this
in-cludes a pump, the pump should be left on overnight For the release of N-linked glycans
incubate at 95°C for 5 h
Trang 86 If the column is not washed with methanol, the final eluent will consist of an immiscible butanol/water mixture, which will prove difficult to dry
7 To ensure that high-purity eluents are always obtained, it is recommended that
ammo-nium acetate be obtained by titrating the relevant acid (e.g., 0.5 M HPLC-grade acetic
acid) to the relevant pH with HPLC-grade ammonium hydroxide Ammonium formate may also be used as an eluent, at similar pH values
8 The precise elution gradient can be varied to suit the diversity of oligosaccharides being studied The pH of the ammonium acetate will greatly affect the resolution and retention and can be tailored to suit the analytes being investigated
9 This flow rate is for a 100 × 4.6 mm Hypercarb S column The smaller Glycosep H col-umn should not be run at flow rates in excess of 0.5 mL/min
10 Great care should be taken to ensure thorough equilibration of the column prior to injecting samples because these columns are quite sensitive to changes in organic-phase composition
11 The use of 0.1% TFA will cause quenching of fluorescence detection
12 To optimize detector sensitivity and avoid baseline drift at low NaOH concentrations, the
addition of 0.3 M NaOH postcolumn (i.e., before it enters the detector) is required to
increase pH to ≥12 Use of a thinner electrode gasket (0.005 in.) gives the best signal-to-noise ratio Flow rate at which the postcolumn reagent is added should be reproducible between runs and must be the gradient pump flow; a pressure of 112–114 psi (eluent vessel must be able to withstand higher pressure) should achieve this flow rate with no significant increase in gradient pump pressure Beware of air bubbles in the beaded
mix-ing coil, which prevent efficient pH increase Note: Always turn off the gradient pump
before turning off the postcolumn eluent stream (20).
13 Retention times will steadily decrease unless the column is regularly regenerated with strong alkali Regenerate the CarboPac PA-100 anion-exchange column for 5 min with
20% 0.5 M sodium acetate – 15 µM NaOH and then re-equilibriate for 30 min with 15 µM
NaOH before each subsequent sample application
14 In addition to introducing 2-AB groups, reductive amination can also be used to introduce
other fluorescent labels, such as 2-aminopyridine (24) or 8-amino naphtalene-1,3,6-trisulfonic acid (25), and also to couple to protein or lipid (see Subheading 1.) Here the
commercial labeling reagent is sufficient to label up tp 50 nmol of oligosaccharide If poor sloubility of the reductant (NaCNBH3) is observed, this can be improved by the addition of 10 µL of water to the labeling mixture prior to adding it to the samples
15 Care should be taken so that the flow rate through the disk is approx 1 drop/s and that air bubbles do not form below the disk Air bubbles can be removed by gentle pressure on the disk; however, it is difficult to remove them completely
16 Desalting may also be carried out by Bio-Gel P2 chromatography eluted in water
Acknowledgment
The author wishes to thank Gail Evans for preparation of the manuscript and the
UK Medical Research Council for funding.
References
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