in-gel o-glycan release in-gel digestion of protein...
TRANSCRIPT
Day 2, O-glycan prep. SDS gel
Excise protein band
Destaining of CBB
Dry gel slices
In-gel O-glycan release In-gel digestion of protein
In-gel reductive b-elimination
Desalt on Dowex H+
Borate removal
O-glycan purification on C18
Permethylation
MS spec.
Reduction and S-carboxyamidomethylation
Trypsin
Peptide extraction
Sampling on C18 packed column
LC-MS/MS analysis
Protein identification by software
Glycan characterization using tools
Morning
Afternoon
Day 1
Permethylation of glycan
Reaction is performed under basic condition
Prepare NaOH-DMSO slurry
CH3I
Biosynthetic pathway of gangliosides
Chapter 10, Figure 2 Essentials of Glycobiology
Second Edition
Biosynthetic pathway of gangliosides
Chapter 10, Figure 2 Essentials of Glycobiology
Second Edition
Biosynthetic pathway of gangliosides
Chapter 10, Figure 2 Essentials of Glycobiology
Second Edition
Biosynthetic pathway of gangliosides
Chapter 10, Figure 2 Essentials of Glycobiology
Second Edition
Biosynthetic pathway of gangliosides
Chapter 10, Figure 2 Essentials of Glycobiology
Second Edition
MS2@660
KA-PJ-32PT-N-glycan-FT-1134-660_090227095111 #1-5 RT: 0.01-0.23 AV: 5 NL: 1.34E4T: FTMS + p NSI Full ms3 [email protected] [email protected] [180.00-2000.00]
200 250 300 350 400 450 500 550 600 650 700
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive A
bundance
618.30
472.21
503.24
415.19
433.20
440.18619.31383.16353.15 473.21 586.28268.11 504.24245.10181.50
605.27
323.14310.31 538.06464.75 630.30390.47 660.31556.32364.24206.66 492.16 679.26
503
415
433
472
MS3@660
660: 1 Hex, 1 HexNAc, 1 deoxyHex.
MS3@660
456
O
O
CH2OMe
NMeAc MeO
329
259
KA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32T: ITMS + c NSI Full ms3 [email protected] [email protected] [180.00-2000.00]
200 250 300 350 400 450 500 550 600 650 700
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive A
bundance
472.29
618.38
586.47
259.22660.45
433.29
503.33
456.32
589.31398.23 630.42329.24 614.32273.01217.95 415.29382.05325.36 367.08 573.53
259
456
415
503
433
433
472
472
329
Sugar modification Sulphate or Phosphate?
Molar mass of H3PO4 = 97.995181 g/mol
Molar mass of H2SO4 = 98.07848 g/mol
Sulfuric acid
Phosphoric acid
S=32.065
P=30.974
Sulfuric acid
Phosphoric acid
Na
CH3
CH3
Glycan backbone
Glycan backbone
Permethylation
M+2Na
M+Na
Desulfation: solvolysis with methanolic HCl
Dephosphorylation: HF treatment
MS2 of PerMe sulfated Lewis A
β-eliminated
Neutral loss of 120
Neutral loss of 120
Neutral loss of 120
Permethylated sulfo Lewisa is enriched in aqueous phase
Permethylation
Mixture of Lea and sulfo-Lea
Phase partition
Extraction of PerMe Glycans
Reconstitute PerMe O-glycan in MeOH
Sample preparation for MS spec.
Add infusion buffer
Positive ion mode
1mM NaOH in 50% MeOH
1mM NaOAc in 50% MeOH
Negative ion mode [M-H]-
MeOH/PrOH/13mM NH4OAc
[M+Na]+
[M+H]+
[M+H]+
[M+Li]+
[M+Na]+
[M+K]+
Full MS profile
Sample analysis by MS spec.
Positive ion mode [M+Na]+
Negative ion mode [M-H]-
Automated MSn analysis
Detect molecular ions of O-glycans
Positive ion mode [M+Na]+
Negative ion mode [M-H]-
Oligosaccharide sequence
Specific epitope
Preparation of base, NaOH-DMSO slurry
Transfer 0.2ml of 50% NaOH in screw top glass tube with P1000
Add 400ul of anhydrous MeOH with pasteur pipette
Vortex
Add 1 pipette full of anhydrous DMSO
Add 1 pipette full of anhydrous DMSO
Spin down
Remove supernatant and insoluble white material.
Repeat ~ 5 times.
Anhydrous
MeOH
50% NaOH
Preparation of base
Transfer 0.2ml of 50% NaOH in screw top glass tube with P1000
Add 400ul of anhydrous MeOH with pasteur pipette
Vortex
Add 1 pipette full of anhydrous DMSO
Add 1 pipette full of anhydrous DMSO
Spin down
Remove supernatant and insoluble white material.
Repeat ~ 5 times.
DMSO & MeOH
NaOH
Preparation of base
Transfer 0.2ml of 50% NaOH in screw top glass tube with P1000
Add 400ul of anhydrous MeOH with pasteur pipette
Vortex
Add 1 pipette full of anhydrous DMSO
Add 1 pipette full of anhydrous DMSO
Spin down
Remove supernatant and insoluble white material.
Repeat ~ 5 times.
Clear
DMSO phase
NaOH slurry
White stuff
Add 200ul of anhydrous DMSO
Add 300ul of NaOH-DMSO
Add 100ul of MeI
Vortex for 5min
1. DMSO
Permethylation
2. NaOH-DMSO
3. MeI
Phase partition
Neutralize reaction mixture with 5% AcOH
Remove MeI with N2
Add DCM
Spin down
Vortex
Transfer supernatant into a clean glass tube
Add water
Spin down
Reaction mixture
Repeat ~ 5 times.
Vortex
Phase partition
Neutralize reaction mixture with 5% AcOH
Remove MeI with N2
Add DCM
Spin down
Vortex
Transfer supernatant into a clean glass tube
Add water
Spin down
AcOH
Repeat ~ 5 times.
Vortex
Phase partition
Neutralize reaction mixture with 5% AcOH
Remove MeI with N2
Add DCM
Spin down
Vortex
Transfer supernatant into a clean glass tube
Add water
Spin down
Bubble off MeI with N2
Repeat ~ 5 times.
Vortex
Needle
Phase partition
Neutralize reaction mixture with 5% AcOH
Remove MeI with N2
Add DCM
Spin down
Vortex
Transfer supernatant into a clean glass tube
Add water
Spin down
Add water
DCM phase
Repeat ~ 5 times.
Vortex
Phase partition
Neutralize reaction mixture with 5% AcOH
Remove MeI with N2
Add DCM
Spin down
Vortex
Transfer supernatant into a clean glass tube
Add water
Spin down
Water phase
Sulfated O-glycan
DCM phase
Neutral &
Sialylated O-glycans
Repeat ~ 5 times.
Vortex
Permethylated sulfo Lewisa is enriched in aqueous phase
Permethylation
Mixture of Lea and sulfo-Lea
Phase partition
DCM phase
PerMe neutral and sialylated O-glycan
Water phase
PerMe sulfated O-glycan
C18 for water phase
Load sample on C18 sep-pack
Elute sulfated O-glycan with 50% ACN
Equilibrate C18 with ACN and 5% AcOH
Dry under N2 stream
Wash C18 sep-pack with 10 ml of water
Day 3, Data analysis
Proteomics, method, protein identification (Toshi, Mayumi)
Tools for glycan analysis (MT, MP)
Comprehensive quantitative glycomics (non-GAG)
Quantitative analysis with STDs
Cell surface glycans
FOS,
nucleotide sugars
LLO
Glycan biosynthesis
and degradation
Proteomics
Glycoproteomics
Site-mapping
Model: S&PS Neural Crest Cells
derived from
Control and S&P iPS cells
Enzymatic release of N-glycan
PNGaseF (a1,6Fuc)
PNGaseA (a1,3Fuc and a1,6Fuc))
Endoglycosidases
(Endo-H, Endo-M)
Microwave assisted enzyme reaction
a.a. sequence sep-pak
sep-pak
graphite carbon
Chemical release of N-glycan
Anhydrous hydrazine (80oC)
(Hydrazine monohydride 90oC)
Unique sugar sequence, modification
Highly fucosylated N-glycan, etc