Transcript No Slide Title

Characterization of Glycan Structure
Essentials in Glycobiology
June 1, 2004
Brad Hayes
Roles of Glycoprotein-associated Carbohydrates
 Quality Control/folding.
(deglucosylation/reglucosylation)
 Solubility.
 Circulating half-life.
(LH, FSH, vWF, ASGPR)
 Cell-cell interactions.
(lymphocyte homing)
Pharmaceutical Concerns
Regarding Carbohydrates
 Pharmacokinetics:
Influence on receptor binding.
 Pharmacodynamics:
Distribution. Clearance.
 Define product as “well-characterized”.
 Lot-to-lot variability.
 Definition of intellectual property.
Carbohydrate Analysis Offers Unique Challenges
 Branched.
 Synthesis is not “template driven”.
 Alternative linkage positions are possible.
 Alternative anomeric configurations are possible.
 Cell-type specific glycosylation.
 Influence of environmental conditions.
[Glucose] [NH3] pH
 Site-specific glycosylation.
 Microheterogeneity.
Considerations in Designing an Analytical Scheme
Question
Amount of
Resources
Material
Workflow
Purity
Sample Prep
Release of Glycans
Enzyme Digestion
Salts
HPLC
Separation
CE
PAD
Detection
Fluorescence
MS
Analysis of Glycans Still Bound to Proteins
Is the protein of interest glycosylated?
Fluorescent detection of sialic acids (<1pMole).
Monosaccharide composition analysis.
If so, are there N-glycans or O-glycans or both?
Mannose vs. Galactosamine (GalNAc)
What is the contribution to molecular weight?
Monosaccharide composition on a per mg basis.
Compare monosaccharide to amino acid composition.
Composition Analysis
Sialic Acid Determination
Diverse family of molecules.
Humans make antibodies against animal proteins withNeuGc.
Sialylation depends on culture conditions.
Incomplete sialylation associated with increased clearance.
When sample limited:
Is my protein glycosylated?
Is there enough material for monosaccharide analysis?
When sample not limited:
Batch-to-batch variability for recombinant proteins.
Extent of deglycosylation for crystallogrphic analysis.
Assess diversity of sialic acids present.
Sialic Acid Determination
Release with mild acid (2 m HOAc, 80oC, 3 hrs.).
“specific” for sialic acid release
doesn’t remove modifications from NeuAc
Anaylze by HPAEC-PAD
sensitivity of ~200 pMoles
Analyze by RP- HPLC after fluorescent derivatization.
OPD
DMB (~ 1 pMole)
Analyze by GC-MS as heptafluorobutyrate derivative.
Sensitive Sialic Acid Determination
Morimoto et.al. Anal. Chem. 2001
Neu5,9Ac2
Fluorescence
Neu5Gc Neu5Ac
BSM
Sialics
Neu5,7Ac2
Neu5Gc9Ac
Neu5,7(8),9Ac3
Reagent
0
5
10
15
20
25
Time (minutes)
30
35
40
45
Composition Analysis: Neutral and Amino Sugars
Is my protein glycosylated?
Are there N- or O-glycans?
Relative contribution to mass?
What are the likely structures?
Hydrolysis, dry, HPAEC-PAD
50
Fuc
Glc
GlcNH2
40
nC
Standards
GalNH2
Gal
Man/Xyl
30
20
0
5
10
15
Time (minutes)
20
25
Analysis of a Human Protein
Predicted Structures
Data
Monosaccharide
nMoles
Fucose
0
Galactosamine
0.366
Glucosamine
0.03
Galactose
0.317
Glucose
0.672
Mannose/Xylose
0.115
NeuAc
0.558
Major Species
Minor Species
a3
b3
a6
a6
b3
b6
No N-Glycans.
On average, only one O-glycan per protein molecule.
No NeuGc, as expected.
Composition Analysis: Neutral and Amino Sugars
GC-MS of TMS derivatives:
or alditol acetates
or heptafluorobutyrate
www.ccrc.uga.edu/~rcarlson/Carbstr.pdf
CE analysis of APTS derivatized
monosaccharides
From Beckman-Coulter Primer 8 on CE
Chen and Evangelista Anal. Chem. 1995
Release of Glycans for Further Analysis
Release of N-glycans:
PNGase F. Broad spectrum for release of N-glycans
blocked by core a1,3 Fuc or bisecting Xyl
PNGase A. core a1,3 Fuc or bisecting Xyl okay
Prefers smaller, neutral glycans
Endo H. Only if ManII has not yet acted
Hydrazine
Release of O-glycans:
Alkaline induced b-elimination. Can’t fluorescently tag
O-glycanase. Only Galb1,3GalNAc
Hydrazine
Release of glycolipid glycans:
Endoglycoceramidase
Glycan Profiling
Charge
Size
Heterogeneity
Comparative differences between samples
Need to consider separation with detection
Profiling Glycans by HPAEC-PAD
Advantages:
Fast.
Few pMole sensitivity.
Reproducible.
N eu5Aca3Galb4GlcNA cb2Mana6
28.85 29.35
N eu5Aca6Galb4GlcNA cb2Mana3
N eu5Aca3Galb4GlcNA cb4
nC
20
Disadvantages:
Retention time only.
Introduces salt.
Sialic acid modifications.
Ma nb4GlcNA cb4GlcNA c(-ol)
10
0
20
N eu5Aca6Galb4GlcNA cb2Mana6
30
Time (minutes)
40
N eu5Aca6Galb4GlcNA cb2Mana3
N eu5Aca3Galb4GlcNA cb4
Ma nb4GlcNA cb4GlcNA c(-ol)
NeuAc(a2,3)Gal elute later than NeuAc(a2,6)Gal
Gal(b1,3)GlcNAc elute later than Gal (b1,4)GlcNAc
Fucosylated elute earlier than non-fucosylated
Neu5Gc elute later than Neu5Ac
+/- Exoglycosidases
Fluorescent Derivatization
Anumula, K. Anal. Biochem. 2000
Profiling/Sequencing Tagged Glycans by NP-HPLC
Anumula and Dhume Glycobiology 1998
Guile et.al Anal. Biochem. 1996
Profiling/Sequencing Glycans by CE-LIF
+Hexosaminidase
+Hexosaminidase
+ 1-2,3 mannosidase
Ma and Nashebeh Anal. Chem. 1990
Profiling Glycans by MALDI-TOF
Mass Spectrometry
Native glycans
Charge heterogeneity
Loss of sialic acid
matrix acidity
Post-source decay
Fragmentation mainly at the glycosidic bonds
Preferential cleavages limit structural information
Permethylated glycans
“Neutralize” carboxyl groups
Reduced desialylation
Enhanced cross-ring cleavages
Simplified Schematic of a MALDI-TOF
Flight tube
High voltage
•
Pulsed laser
With MALDI ionization get almost
exclusively singly charged species
MALDI-TOF Profiling of Glycans in Disease
Normal
or
10,000
4414.83
2793.05
A
B
U
N
D
A
N
C
E
5,000
4589.03
2431.68
3242.52
3603.90
4053.39
3692.02
3487.79
4228.86
4660.12
4834.30
5324.88
5572.17
5151.69
Patient
8,000
2431.87
4,000
2793.05
3243.29
2000
2800
4053.72
3692.49
3866.59 4227.81
4414.83
3603.48
3937.64
3600
4400
MASS (m/z)
5324.38
5151.25
5571.77
5200
6000
Sequencing of Glycans with Mass Spectrometry
Already touched on use of exoglycosidases with
separation either by HPLC or CE
and detection either by PAD or fluorescence
Can also use mass spectrometry for sequencing.
Mechref et.al. Carbo Res. 1998
Branching and Linkage Analysis
How the components are put together
Sequencing of Glycans with Mass Spectrometry
Mechref et.al. Carbo Res. 1998
Linkage Analysis with Mass Spectrometry
Ionization
Mass Analyzer/
Collision
Method
Selector
Cell
MALDI
Quadrapole
ESI
Ion Trap
Detector
Quadrapole
Ion Trap
TOF
Linkage Analysis with Mass Spectrometry
Sheeley and Reinhold Anal. Chem. 1998
Linkage analysis by GC-MS of partially
methylated alditol acetates.
Methylate exposed hydroxyl groups.
Hydrolyze glycosidic bonds.
Reduce with borohydride.
Acetylate newly created hydroxyl groups.
Analyze by GC-MS.
Linkage Analysis of Total N-Glycans from
ST6Gal-I Deificient Mice by GC-MS
Abundance
ST6Gal-I wt
3Gal1
Gal1-
Abundance
14
16
18
6Gal1
20
22
time (minutes)
24
ST6Gal-I null
2Man1
2,4Man1
2,6Man1
3,6Man1
Fuc114
16
18
20
time (minutes)
22
24
Linkage Analysis by NMR Spectroscopy
Sia3
H3eq Sia6
H3eq
(–)
Sia3
H3ax Sia6
H3ax
(+)
500 MHz nano-NMR spectra from total N-glycans
from ST6Gal-I deficient mice .
MALDI-TOF Analysis of HPAEC Fractions
400
2
300
N-Glycans from ST6Gal-I
deficient mouse liver
16 17
1
14
12
200
44
nC
45
100
4
5
31
10
9
0
0
A
B 7000
U
N
D
A
N
0
C
2000
E
20
10
43
20
30
46
40
50
60
Time (minutes)
2853.10
2600
3200
6000
Peak 44
3800
MASS (m/z)
4400
5000
2000
2853.10
2600
Peak 45
3200
3800
MASS (m/z)
4400
5000
Structure of Sulfated Glycosaminoglycans
Analysis of Glycosaminoglycans
Isolate GAG/proteoglycan by ion exchange
chromatography.
Depolymerize enzymatically to generate disaccharides.
Fraction disaccharides (IP-RP HPLC, IEC HPLC).
Detect by UV (>100 pMoles).
Detect fluorescently following post-column
derivatization (<10 pMoles).
Post-column Labeleing with 2-Cyanoacetamide
 2-Cyanoactamide reacts with reducing sugars under basic
conditions at high temperature.
 Used as post-column derivatization for GAG disaccharide
analysis.
 Applicable to any separation.
Flow Path for Post-column Labeling
>100 pMole
UV Detector
Column
IP-RP HPLC
IEC HPLC
1%
2-CA
0.25 M
NaOH
Fluorescence
Detector
>5 pMole
“Cooling
Bath”
Reaction
Coil
130oC
Ion-Pair Rerverse Phase HPLC
Analysis of Serum Chondroitin Sulfate
800
Control
Di-4S
600
Fluorescence
400
200
Di-0S
200
Patient
Di-4S
Di-0S
100
Di-6S
Di-diSE
0
Di-0S
Di-UA2S
Di-6S
Di-4S
300
Di-diSB
Stds
Di-diSD
Di-diSE
100
C h on droi ti n ase Dis acch aride s
Re al Name
S h ort From
UA-GalNAc
Di-0S
UA-GalNAc-4S
Di-4S
UA-GalNAc-6S
UA-GalNAc-4S-6S
UA-2S-GalNAc
UA-2S-GalNAc-4S
UA-2S-GalNAc-6S
UA-2S-GalNAc
Di-TriS
0
0
10
20
30
40
50
Time (minutes)
60
70
Results from ~10 ml of serum
Di-6S
Di-diSE
Di-UA2S
Di-diSb
Di-diSd
Di-t riS
Ion Exchange Fractionation of
Heparin-derived Disaccharides
UA-[1,4]-GlcNAc
200
UA-[1,4]-GlcNS
UA-[1,4]-GlcNAc-6S
UA-2S-[1,4]-GlcN
UA-2S-[1,4]-GlcN-6S
UA-[1,4]-GlcN-6S
100
50 pMole of Heparin Disaccharides Stds
UA-2S-[1,4]-GlcNAc-6S
UA-2S-[1,4]-GlcNS-6S
UA-[1,4]-GlcNS-6S
UA-[1,4]-GlcN
Fluorescence
UA-2S-[1,4]-GlcNS
0
1000
Lyase Digest of 1 ug Authentic Heparin
UA-2S-[1,4]-GlcNS-6S
UA-[1,4]-GlcNAc-6S
UA-[1,4]-GlcNS
UA-2S-[1,4]-GlcNAc-6S
UA-[1,4]-GlcNS-6S
UA-2S-[1,4]-GlcNS
UA-[1,4]-GlcNAc
0
0
10
20
30
40
Time (minutes)
50
60
70
Test for O-GlcNAc
O-GlcNAc is a substrate for galactosyltransferase and can
be radiolabeled using UDP-[3H]galactose as the donor.
b1,4 Galactosyltransferase
Protein-O-GlcNAc
Protein-O-GlcNAc-[3H]Gal
UDP-[3H]Gal UDP
O-GlcNAc is susceptible to alkaline-induced b-elimination.
Protein-O-GlcNAc-[3H]Gal
NaOH
NaBH4
[3H]Galb1,4GlcNAcitol
Galb1,4GlcNAcitol
Galb1,3GalNAcitol
Galb1,3GlcNAcitol
Pit 1 is O-GlcNAcylated
Separation of disaccharide alditols on Dionex MA-1 column
Summary
There are many options available for characterizing glycans.
The choice of approaches and technologies depends on
the question asked and the depth of
understanding required.
A complete structural characterization generally
requires the use of several different and
complementray techniques.