Monosaccharide, Oligosaccharide,
and Linkage Analysis
Natasha E. Zachara Ph.D.
nzachara@jhmi.edu
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What questions might you ask?
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AcNH
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• What monosaccharides modify my protein?
• What oligosaccharides modify my protein?
• How are these sugars linked to each other?
As always, the choices you make about how you will approach
answering these questions will depend in part on the equipment
available to you.
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AcNH
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Monosaccharide Analysis
• Why?
– Knowing which sugars helps to predict the type of
oligosaccharides present and thus helps determine the
approach to detailed structural analysis
– It can also provide the first clues to the presence of a new type
of sugar chain.
• Step one: release of sugars
• Step two: labeling of sugars (if necessary)
• Step three: detection and quantification
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Sample Preparation
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AcNH
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• In most cases, salt is an issue:
• Proteins can be precipitated to remove salt
• Acetone
• Methanol
• Methanol/Chloroform
• Protein can be desalted by Size Exclusion Chromatography
• Ammonium formate, ammonium bicarbonate (volatile)
• Sephadex-G50, many others
• Proteins/peptides purified by reversed phase are also acceptable
• Proteins can be separated by SDS-PAGE and blotted to PVDF
• Sugars can be desalted
• Size exclusion chromatography (time challenging)
• Dowex Ion Exchange (in a volatile buffer)
• Graphitized Carbon
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Release: monosaccharides
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AcNH
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Acid hydrolysis:
• Different sugars and linkages are released at different rates
• Low acid: Sialic acid
• Neutral and amino sugars: 2M TFA and 4M HCl
Harazono A, et al., A comparative study of monosaccharide composition analysis as a carbohydrate test for
biopharmaceuticals. Biologicals. 2011 May;39(3):171-80. PMID: 21549615
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Detection
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AcNH
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• The challenge is detecting your released sugars:
• PAD: Pulsed Ampometric Detection (no labeling)
• Metabolic Labeling
• Derivatization for HPLC:
• 2-aminopyridine (2AP),
• ethyl 4-aminobenzoate (ABEE),
• 2-aminobenzoic acid (2AA)
• 1-phenyl-3-methyl-5-pyr- azolone (PMP)
• 1,2-diamino-4,5-methylenedioxybenzene (DMB)
• Capillary Electrophoresis:
• 8-aminopyrene-1,3,6-trisulfonate (APTS)
• 2-aminoacridone derivatization
• Derivatization for GC-MS:
• trime- thylsilyl or alditol acetate
Harazono A, et al., A comparative study of monosaccharide composition analysis as a carbohydrate test for
biopharmaceuticals. Biologicals. 2011 May;39(3):171-80. PMID: 21549615
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Separation
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AcNH
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• HPLC
– Anion Exchange Chromatography (HPAEC)
– Reversed Phase
– Ion Exchange (TSK Fractogel)
• Capillary Electrophoresis
• GC-MS
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AcNH
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High Performance Anion Exchange ChromatographyPulsed Ampometric Detection (HPAEC-PAD)
• Advantages:
• No Labeling
• No interference from labeling reagent
• Reasonable sensitivity (10pmol)
• Disadvantages:
• Specialized HPLC (All hydroxide)
• The Separation:
• Under alkaline conditions, the hydroxyl groups
are ionized to oxyanions
• A monosaccharide possesses several
ionizable hydroxyl groups with the following
hierarchy of acidity:
1-OH > 2-OH >> 6-OH > 3-OH > 4-OH
• The varying location of the OH groups results
in slight differences in the pKa value (ranging
from 12 to 14) of individual monosaccharides
Harazono A, et al., A comparative study of monosaccharide composition analysis as a carbohydrate test for
biopharmaceuticals. Biologicals. 2011 May;39(3):171-80. PMID: 21549615
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HPAEC-PAD Columns
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AcNH
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CarboPAc PA1, 10, 20
• Monosaccharides
• Disaccharides
• Sialic acid
CarboPAc PA 100, 200
• Oligosaccharides
CarboPAc MA1
• Aliditols (monosaccahrides, oligosaccharides)
Behan JL, Smith KD. The analysis of glycosylation: a continued need for high pH anion exchange chromatography. Biomed Chromatogr. 2011
Jan;25(1-2):39-46. PMID: 20821735
How does pad work?
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AcNH
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Ampometric Detection:
• Any analyte that can be oxidized or reduced is a
candidate for amperometric detection
• A voltage (potential) is applied between two
electrodes positioned in the column effluent
• The measured current changes as an
electroactive analyte is oxidized at the anode.
Pulsed Ampometric Detection:
• Important as it ensures that the gold electrode
does not become coated by oxides
• In pulsed amperometric detection (PAD), a
working potential is applied for a short time
(usually a few hundred milliseconds), followed by
higher or lower potentials that are used for
cleaning the electrode
• The detection of monosaccharides and aminecontaining glycoconjugates with low levels of
glycosylation can be compromised from fouling
of the working electrode by amino acids.
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Examples: monosaccharides
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AcNH
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http://www.dionex.com/
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Examples: monosaccharides
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AcNH
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Note, that Sodium Acetate is required to elute sugar phosphates
http://www.dionex.com/
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Sugar Alditols
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AcNH
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Note, that a gradient of sodium hydroxide is required
http://www.dionex.com/
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HPAEC: SIALIC acid
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AcNH
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The strong charge requires more robust conditions; an increasing
sodium acetate linear gradient is useful (50–250 mm over 30 min .
O-Acetyl groups can be labile, andcan saponify during analysis.
There are alternative non-high pH based methods.
http://www.dionex.com/
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HPAEC: Neutral oligosaccharides
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AcNH
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• Separation of oligosaccharide is based on size, charge, monosaccharide composition
and intra-chain linkages
• Separation is based on a sodium acetate gradient; the greater the negative charge
(greater the number of NeuAc) and number of sugars, the higher the sodium acetate
concentration required
http://www.dionex.com/
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HPAEC: Charged Oligosaccharides
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AcNH
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the greater the overall negative charge, the greater the
retention time.
http://www.dionex.com/
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Desalting post-HPAEC
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AcNH
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Packer NH, Lawson MA, Jardine DR, Redmond JW. Glycoconj J. 1998 Aug;15(8):737-47. A general approach to desalting
oligosaccharides released from glycoproteins. PMID: 9870349
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GC-MS: monosaccharides
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AcNH
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Sugars must be derivatized: trimethyl esters, aldlitol acetates, permethylation
Also possible to release sugars by methanolysis, and derivatize to TMS derivatives
Detection: Flame Ionization detector
Several types of stationary phases can be used for the analysis of acetylated alditols:
• CP-Sil 5 WCOT, SP-1000, Silar 10C, OV-1, SE-54, 5% DB-5, DB-1, Carbowax 20M, SE-30,
OV-101, and OV-275.
• Other stationary phases used for the analysis of trimethylsilylated glycosides and methyl
glycoside methyl esters include CP-Sil 5 WCOT, 5% DB-5, DB-1, SE-30, and OV-101.
PMID: 6057635
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GC-MS: monosaccharides
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AcNH
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• Identify sugars by retention time and fragmentation patterns
• Lose some information re: modifications of sugars
• Quantitative
PMID: 6057635
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HPLC: monosaccharides
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AcNH
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Labeling with different derivatives:
A: 2AP (2-aminopyridine )
B: ABEE (ethyl 4-aminobenzoate )
C: 2AA (2-aminobenzoic acid )
D: PMP (1-phenyl-3-methyl-5-pyrazolone
)
Separation Technologies:
Strong Anion Exchange (TSK Fractogel)
Reversed Phase (C18)
CE
Great sensitivity…
Derivatization can fluctuate in efficiency
May be necessary to include a clean up
step
Longer run times
Harazono A, et al., A comparative study of monosaccharide composition analysis as a carbohydrate test for
biopharmaceuticals. Biologicals. 2011 May;39(3):171-80. PMID: 21549615
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Capillary Electrophoresis (CE)
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AcNH
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Separation buffers: 50 mM sodium
phosphate (pH 5.5) or 150 mM sodium
borate-50 mM sodium phosphate (pH
7.0) running buffer.
Run times are fast
Compatible with mass spectrometry
Not preperative
Abo M, He LP, Sato K, Okubo A. Determination of monosaccharides derivatized with 2-aminobenzoic Acid by capillary
electrophoresis. Methods Mol Biol. 2013;984:45-50. doi: 10.1007/978-1-62703-296-4_4. PMID:23386335
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Oligosaccharides
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AcNH
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• There are three steps:
• Step one: Release
• Step two: Detections
• Step three: Separation
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Release: oligosaccharides
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AcNH
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– Reductive b-elimination:
• releases O-linked sugars, but the carbohydrates must be reduced to prevent
peeling.
• results in sugar alditols, which are not compatible with many labeling
techniques
• Alditol acetates are volatile and are easily detected by GC-MS.
– Hydrazinolysis:
• Hydrazine hydrolysis is an effective method for the complete release of
unreduced O- and N-linked oligosaccharides.
• not as popular as other techniques as it’s toxic and can affect the integrity of
the peptide/protein backbone.
– Enzyme hydrolysis:
• PNGase F releases most N-linked glycans.
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Detection: Oligosaccharides
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AcNH
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• Radiolabels:
• Metabolic Labeling
• Glycosyltransferases
 b-elimination
• Reductive amination:
• Incorporation of fluoresecent derivatives (see above)
• Pulsed amometric detection
• Mass spectrometry (GC, MALDI, ESI-MS)
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Separation
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AcNH
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Thin Layer Chromatography
High-voltage Borate Paper electrophoresis
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Size exclusion/Gel Permeation Chromatography:
HPLC
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Graphitized Carbon
Reversed Phase
HILIC
HPAEC
• GC-MS
• Mass Spectrometry
• Can be combined with HPLC
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HPLC: Graphitized Carbon
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AcNH
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• Not entirely clear how graphitized
carbon separates oligosaccharides,
although size, shape and charge
are all important
• Can separate anomers
• N-linked sugars should be reduced
to avoid peak splitting
• Sensitive to the low femtomole level
• Columns require significant
regeneration
Karlsson NG, Wilson NL, Wirth HJ, Dawes P, Joshi H, Packer NH. Rapid Commun Mass Spectrom. 2004;18(19):2282-92.
Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein
oligosaccharide analysis.
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AcNH
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HPLC: hydrophilic interaction liquid
chromatography (HILIC)
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HILIC: Sugars are separated by
a partitioning mechanism in
contrast to the traditional
adsorption chromatography on
normal phase materials
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Typical equilibration conditions
10–25% water in acetonitrile
with a low concentration of
acid or salt (mostly below 100
mM)
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Common columns include: TSK
Gel-Amide 80 and ZIC-HILIC-SPE
•
Compatible with spectrometry
•
Detection requires
derivatization
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Biogel p4
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AcNH
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• Carbohydrates can be separated
by size
• Typically Biogel P-2, P-4 and P-6
(<400 mesh resin) are used.
• Biogel P-4 is best for the
separation of 3-24 units
• Sugars are detected by the
incorporation of a radiolabel or a
fluorescent tag
• For O-linked/N-linked sugars it is
possible to introduce this tag
during reductive b-elimination, by
using tritiated sodium borohydride
• The carbohydrates can also be
detected using colorimetric
assays
Columns are calibrated and
retention time expressed in
units of glucose.
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FACE: fluorophore-assisted carbohydrate electrophoresis
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AcNH
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• Separate oligosaccharides by
electrophoresis
• Addition of a tag (by reductive
amination) provides a charge
and a meads of detection
• Separation may vary if sugars
are sulfated, acetylated of
sialylated as this will change
the size: charge ratio.
• Can be combined with
enzyme treatments to look at
the structure of the
oligosaccharides, however,
this requires purified oligo’s
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GC-MS: Oligosaccharides
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AcNH
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• Large oligosaccharides (>10) are difficult to detect
• Must use alternative approaches, eg. FAB-MS
• Typically permethylated
Karlsson N G et al. J. Biol. Chem. 1997;272:27025-27034
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Fragmentation: Oligosaccharides
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AcNH
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Karlsson N G et al. J. Biol. Chem. 1997;272:27025-27034
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Linkage Analysis
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AcNH
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• What questions are you really asking?
• Which sugars are linked to each other, and in what order
• Though which hydroxyl residues are these sugars linked
• What is the anomericity of the linkage?
• Methods:
• NMR (best, but requires largish amounts of pure material)
• Exoglycosidase digestion of oligosaccharides (requires the right enzymes)
• Methylation Analysis:
• GC-MS
• ESI-MS (best with upstream separation of oligosaccharides)
• You may get some information about anomericity when this is performed in
tandem with column chromatography
• MALDI-TOF
• Mass Spectrometry
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Methylation Analysis
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AcNH
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Methylation analysis is a well-established method for
determining linkage positions.
1) Introduce a stable substituent (an ether-linked
methyl group) onto each free hydroxyl group of
the native glycan
2) Acid cleavage (glycosidic linkages and more labile
than ether-linked methyl groups)
3) Partially methylated monosaccharides with free
hydroxyl groups are derivatized to produce
volatile molecules amenable to GLC-MS analysis.
4) Typically, monosaccharides are reduced (no alpha
or beta anomers) followed by acetylation of the
free hydroxyls
5) Detection by GC-MS or ESI-MS
No sequence information, no anomeric linkage
information
Essentials of Glycobiology
Second Edition
Chapter 47, Figure 3
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AcNH
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Methylation Also Improves Assignment During
Mass Spectrometry
What Mass Spectrometry can do:
1) heterogeneity and type of glycosylation;
2) sites of glycosylation;
3) glycan-branching patterns;
4) the number and lengths of antennae, their building
blocks, and the patterns of substitution with
fucose, sialic acids, or other capping groups such
as sulfate, phosphate, or acetyl esters;
5) complete sequences of individual glycans.
What it can’t do:
Which Glucose versus Mannose
Anomericity
How does Methylation Help:
Better data
Greater confidence in linkage analysis by MS
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Wednesday
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AcNH
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• This will be a demonstration laboratory:
– You will set up one protein for sequencing (and analyze the cycles)
– We will talk about strategies for site-mapping proteins (mini-lecture)
– You will analyze some spectra of O-GlcNAc modified peptides
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Reading
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AcNH
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•
Harazono A, et al., A comparative study of monosaccharide composition analysis as a carbohydrate test for
biopharmaceuticals. Biologicals. 2011 May;39(3):171-80. PMID: 21549615
•
Behan JL, Smith KD. The analysis of glycosylation: a continued need for high pH anion exchange
chromatography. Biomed Chromatogr. 2011 Jan;25(1-2):39-46. PMID: 20821735
•
Manzi AE. 2001 May;Chapter 17:Unit17.19A. Total compositional analysis by high-performance liquid
chromatography or gas-liquid chromatography. PMID: 18265151
•
Manzi AE, Diaz S, Varki A. Anal Biochem. 1990 Jul;188(1):20-32. High-pressure liquid chromatography of sialic
acids on a pellicular resin anion-exchange column with pulsed amperometric detection: a comparison with
six other systems. PMID: 2221361
•
Reinhold V, Zhang H, Hanneman A, Ashline D. Mol Cell Proteomics. Toward a platform for comprehensive
glycan sequencing. 2013 Apr;12(4):866-73. PMID: 23438731
•
Hansson GC, Karlsson H. Methods Mol Biol. Gas chromatography and gas chromatography-mass
spectrometry of glycoprotein oligosaccharides. 1993;14:47-54. PMID: 8348243
•
Kim JH, Shome B, Liao TH, Pierce JG. Anal Biochem. Analysis of neutral sugars by gas-liquid chromatography
of alditol acetates: application to thyrotropic hormone and other glycoproteins. 1967 Aug;20(2):258-74. PMID:
6057635