Monosaccharide, Oligosaccharide, and Linkage Analysis Natasha
E. Zachara Ph.D. [email protected]
Slide 2
O O HO AcNH What questions might you ask? 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.
Slide 3
O O HO AcNH 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 Monosaccharide Analysis
Slide 4
O O HO AcNH Sample Preparation 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
Slide 5
O O HO AcNH Release: monosaccharides 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
AHarazono A, et al., A comparative study of monosaccharide
composition analysis as a carbohydrate test for biopharmaceuticals.
Biologicals. 2011 May;39(3):171-80. PMID: 21549615Biologicals.
Slide 6
O O HO AcNH Detection 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 AHarazono A, et al., A
comparative study of monosaccharide composition analysis as a
carbohydrate test for biopharmaceuticals. Biologicals. 2011
May;39(3):171-80. PMID: 21549615Biologicals.
Slide 7
O O HO AcNH Separation HPLC Anion Exchange Chromatography
(HPAEC) Reversed Phase Ion Exchange (TSK Fractogel) Capillary
Electrophoresis GC-MS
Slide 8
O O HO AcNH High Performance Anion Exchange Chromatography-
Pulsed 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 pK a value (ranging
from 12 to 14) of individual monosaccharides Harazono AHarazono A,
et al., A comparative study of monosaccharide composition analysis
as a carbohydrate test for biopharmaceuticals. Biologicals. 2011
May;39(3):171-80. PMID: 21549615Biologicals.
Slide 9
O O HO AcNH HPAEC-PAD Columns Behan JLBehan 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: 20821735Smith KDBiomed Chromatogr. CarboPAc PA1, 10, 20
Monosaccharides Disaccharides Sialic acid CarboPAc PA 100, 200
Oligosaccharides CarboPAc MA1 Aliditols (monosaccahrides,
oligosaccharides)
Slide 10
O O HO AcNH How does pad work? 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 amine- containing glycoconjugates with low
levels of glycosylation can be compromised from fouling of the
working electrode by amino acids.
Slide 11
O O HO AcNH Examples: monosaccharides
http://www.dionex.com/
Slide 12
O O HO AcNH Examples: monosaccharides http://www.dionex.com/
Note, that Sodium Acetate is required to elute sugar
phosphates
Slide 13
O O HO AcNH Sugar Alditols Note, that a gradient of sodium
hydroxide is required http://www.dionex.com/
Slide 14
O O HO AcNH HPAEC: SIALIC acid The strong charge requires more
robust conditions; an increasing sodium acetate linear gradient is
useful (50250 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/
Slide 15
O O HO AcNH HPAEC: Neutral oligosaccharides 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/
Slide 16
O O HO AcNH HPAEC: Charged Oligosaccharides the greater the
overall negative charge, the greater the retention time.
http://www.dionex.com/
Slide 17
O O HO AcNH Desalting post-HPAEC Packer NHPacker 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: 9870349Lawson MAJardine DRRedmond JWGlycoconj
J.
Slide 18
O O HO AcNH GC-MS: monosaccharides 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
Slide 19
O O HO AcNH GC-MS: monosaccharides Identify sugars by retention
time and fragmentation patterns Lose some information re:
modifications of sugars Quantitative PMID: 6057635
Slide 20
O O HO AcNH HPLC: monosaccharides 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
AHarazono A, et al., A comparative study of monosaccharide
composition analysis as a carbohydrate test for biopharmaceuticals.
Biologicals. 2011 May;39(3):171-80. PMID: 21549615Biologicals.
Slide 21
O O HO AcNH Capillary Electrophoresis (CE) 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
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
Slide 22
O O HO AcNH Oligosaccharides There are three steps: Step one:
Release Step two: Detections Step three: Separation
Slide 23
O O HO AcNH Release: oligosaccharides Reductive -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 its toxic and can affect the
integrity of the peptide/protein backbone. Enzyme hydrolysis:
PNGase F releases most N-linked glycans.
Slide 24
O O HO AcNH Detection: Oligosaccharides Radiolabels: Metabolic
Labeling Glycosyltransferases -elimination Reductive amination:
Incorporation of fluoresecent derivatives (see above) Pulsed
amometric detection Mass spectrometry (GC, MALDI, ESI-MS)
Slide 25
O O HO AcNH Separation Thin Layer Chromatography High-voltage
Borate Paper electrophoresis FACE Size exclusion/Gel Permeation
Chromatography: HPLC Graphitized Carbon Reversed Phase HILIC HPAEC
GC-MS Mass Spectrometry Can be combined with HPLC
Slide 26
O O HO AcNH HPLC: Graphitized Carbon 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.
Slide 27
O O HO AcNH HPLC: hydrophilic interaction liquid chromatography
(HILIC) HILIC: Sugars are separated by a partitioning mechanism in
contrast to the traditional adsorption chromatography on normal
phase materials Typical equilibration conditions 1025% water in
acetonitrile with a low concentration of acid or salt (mostly below
100 mM) Common columns include: TSK Gel-Amide 80 and ZIC-HILIC-SPE
Compatible with spectrometry Detection requires derivatization
Slide 28
O O HO AcNH Biogel p4 Carbohydrates can be separated by size
Typically Biogel P-2, P-4 and P-6 (
Slide 29
O O HO AcNH FACE: fluorophore-assisted carbohydrate
electrophoresis 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
oligos
Slide 30
O O HO AcNH GC-MS: Oligosaccharides 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
Slide 31
O O HO AcNH Fragmentation: Oligosaccharides Karlsson N G et al.
J. Biol. Chem. 1997;272:27025-27034
Slide 32
O O HO AcNH Linkage Analysis 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
Slide 33
O O HO AcNH Methylation Analysis Chapter 47, Figure 3
Essentials of Glycobiology Second Edition 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
Slide 34
O O HO AcNH 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 cant do: Which Glucose versus Mannose Anomericity
How does Methylation Help: Better data Greater confidence in
linkage analysis by MS
Slide 35
O O HO AcNH Wednesday 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
Slide 36
O O HO AcNH Reading 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 Harazono ABiologicals. 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 Behan JLSmith KDBiomed Chromatogr. Manzi AE. 2001
May;Chapter 17:Unit17.19A. Total compositional analysis by
high-performance liquid chromatography or gas-liquid
chromatography. PMID: 18265151 Manzi AE 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 Manzi AEDiaz SVarki AAnal Biochem.
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 Reinhold VZhang HHanneman AAshline
DMol Cell Proteomics. Hansson GC, Karlsson H. Methods Mol Biol. Gas
chromatography and gas chromatography-mass spectrometry of
glycoprotein oligosaccharides. 1993;14:47-54. PMID: 8348243 Hansson
GCKarlsson HMethods Mol Biol. 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 Kim JHShome BLiao THPierce JG Anal Biochem.