Glycomics Profile Analysis by
MALDI TOF/MS in Human CSF
Xueli Li, Ph.D.,
Children’s Hospital of
Philadelphia
Children’s Hospital of Philadelphia
Introduction
• Genetic defects in human glycome
– 2-3% of the human genome encodes proteins for
glycosylation
– 50% of proteins are glycosylated in human
– 10 distinct glycoprotein biosynthesis pathways
– >100 disease causing genes discovered
– Many still remain unknown
Children’s Hospital of Philadelphia
Why CSF glycome?
• Glycoproteome and glycome in plasma are limited
• Unique glycosylation type and glycogene expression
pattern in brain
• CSF glycome in human has not been well studied
• Does CSF glycome provide us more diagnostic value
for CDG ?
Children’s Hospital of Philadelphia
Project
•
•
•
•
CSF free glycome
CSF N-glycome
CSF O-glycome
Study cohort
– 166 CSF from patients with undiagnosed
neurological disease (NIH/UDP)
– 10 CSF normal controls
Children’s Hospital of Philadelphia
Methods
400 μl CSF
Filter
Glycoprotein
Free glycan
N-Glycan
PNGase F
O-Glycan
Reduced β-elimination
Desalted & Purified
by C18 column
Desalted & purified by
acetic acid – methanol
& resin
Permethylation
Permethylation
MALDI-TOF
MALDI-TOF
Children’s Hospital of Philadelphia
“Undiagnosed” Cases
Brother (MM, 11 year old)
Encephalopathy/CP/Spast
ic Paraparesis
Congenital Hypotonia
Global Developmental
Delay
Bilateral Hearing Loss
Multiple Fractures
Recurrent Pneumonias
Low immunoglobulin
level, low IgA
Generalized
Aminoaciduria
CDG normal by CDT
Sister (IM, 6 year old)
Profound Global
Developmental Delay
Congenital Hypotonia
Neonatal Seizures
Cerebral atrophy, small
corpus callosum
History of recurrent UTIs
CDG normal by CDT
Children’s Hospital of Philadelphia
838.57
Sib’s Urine Free Glycan
48000
885.4
20000
Glu4 or Glu3Man1
patient
681.57
0
1742.86
Intensity
24000
1241.30
5000
885.92
1275.35
10000
Control
651.68
Intensity
15000
0
1000
1500
2000
m/z
2500
3000
800
1600
2400
3200
4000
m/z
Children’s Hospital of Philadelphia
Differential For Hex4
Pompe Disease
acid alpha-1,4glucosidase (GAA),
17q25.2-q25.3
CDG IIb
α1,2
glucosidase I (GCS1),
2p13-p12
Glucosidase I
X
Polyglucose Diet
Glc(3)-Man(9)-GlcNAc(2)
Children’s Hospital of Philadelphia
Sib’s CSF Free Glycan
Control
Patient
Children’s Hospital of Philadelphia
Abnormal CSF Free Glycans
m/z
value
Reference
range
885.40
Hex4 (Glu4 or Glu3Man1)
43.21%
H
1.37-8.59%
1538.697
Man6 GlcNAc1 or Glu3Man3GlcNAc1
0.41%
H
0.0-0.13%
1742.86
Man7 GlcNAc1 or Glu3Man4GlcNAc1
17.07%
H
0.0-0.0%
1946.96
Man8 GlcNAc1 or Glu3Man5GlcNAc1
0.32%
H
0.0-0.0%
2151.06
Man9 GlcNAc1 or Glu3Man6GlcNAc1
0.14%
H
0.0-0.0%
2355.06
Glu3Man7GlcNAc1
0.10%
H
0.0-0.0%
Children’s Hospital of Philadelphia
Children’s Hospital of Philadelphia
Freeze Nature Reviews Genetics advance online publication; published online 6 June 2006 | doi:10.1038/nrg1894
10
1241.4728
1601.8
2204.6
Mass (m/z)
2807.4
Children’s Hospital of Philadelphia
3410.2
3950.5535
3833.4614
3776.4980
3731.4783
3690.3521
3648.4072
3602.4058
3541.3098
3415.3765
3356.3542
3283.2417
3241.2546
3140.2556
3047.3540
2966.1733
3002.2876
2882.1362
2430.9685
2792.0989
90
2778.0840
2788.0940
2834.0967 2822.1096
2738.1023
2686.2212
2600.0334
2546.9954
2506.9702
2460.9790
30
2416.9443
2359.9280
2302.8977
100
2226.9016
2185.8855
2069.8403
2028.8046
1981.8041
1925.8187
1866.7703
1823.6553
1783.7192
1742.6985
1701.6742
1661.6952
1620.6633
1553.0438
1416.5986
1293.5281
20
1579.6453
1118.5491
1175.5707
0
999.0
1053.4089
1000.4803
% Intensity
Sib’s Plasma N-glycan
4700 Reflector Spec #1 MC[BP = 2793.1, 11203]
12-17-2010-10BN09688
Glc3Man7
1.1E+4
80
70
60
50
40
4013.0
Sib’s CSF N-Glycan Compare with Control
Patient CSF N-Glycan
3009.22
Wrong slides
2805.08
Control CSF N-Glycan
Children’s Hospital of Philadelphia
MOGS Seq
• Affected male sibling
Exon 1 c.65C>A, p.A22E; c.329G>A, p.110R>H;
Exon 2 c.370C>T, p.124Q>X
• Affected female sibling
Exon 1 c.65C>A, p.A22E; c.329G>A, p.110R>H
Exon 2 c.370C>T, p.124Q>X
• Mother
Exon 2 c.370C>T, p.124Q>X
• Father
Exon 1 c.65C>A, p.A22E; c.329G>A, p.110R>H;
Children’s Hospital of Philadelphia
GalNAc Type O-linked Protein Glycosylation
Core1
Biosynthesis of Core 1 and Core 2
Core2
Essentials of Glycobiology
Second Edition
Children’s Hospital of Philadelphia
Plasma O-glycan Profile
1200
Monosialyl T antigen
895.584
Intens. [a.u.]
1000
800
M-core 2
600
Disialyl T
400
1256.790
D-core 2
I.S.
200
*
1391.894
1344.874
0
800
1200
m/z
1705.983
1600
Children’s Hospital of Philadelphia
CSF O-glycan Profiles
• Control and abnormal CSF O-linked glycan
Control
CAD-Def
Children’s Hospital of Philadelphia
CAD Deficiency
Children’s Hospital of Philadelphia
Conclusion
• CSF glycome is different and more complex than
plasma glycome
– Free glycome, N-glycome and O-glycome
• CSF glycome may detect CNS-specific defect in
protein glycosylation
• CSF glycome provides additional information for
the diagnosis of CDG
Children’s Hospital of Philadelphia
Acknowledgment
He Research Lab
Miao He, Ph.D.
Mohd Raihan
NIH/UDP
Mariska Davids, Ph.D.
Megan Kane, Ph.D.
Lynne Wolfe
Neal Boerkoel, MD. Ph.D.
Bill Gahl, MD. Ph.D.
Children’s Hospital of Philadelphia