in-gel o-glycan release in-gel digestion of protein...
TRANSCRIPT
![Page 1: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/1.jpg)
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
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Permethylation of glycan
Reaction is performed under basic condition
Prepare NaOH-DMSO slurry
CH3I
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Biosynthetic pathway of gangliosides
Chapter 10, Figure 2 Essentials of Glycobiology
Second Edition
![Page 4: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/4.jpg)
Biosynthetic pathway of gangliosides
Chapter 10, Figure 2 Essentials of Glycobiology
Second Edition
![Page 5: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/5.jpg)
Biosynthetic pathway of gangliosides
Chapter 10, Figure 2 Essentials of Glycobiology
Second Edition
![Page 6: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/6.jpg)
Biosynthetic pathway of gangliosides
Chapter 10, Figure 2 Essentials of Glycobiology
Second Edition
![Page 7: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/7.jpg)
Biosynthetic pathway of gangliosides
Chapter 10, Figure 2 Essentials of Glycobiology
Second Edition
![Page 8: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/8.jpg)
![Page 9: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/9.jpg)
![Page 10: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/10.jpg)
MS2@660
![Page 11: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/11.jpg)
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
![Page 12: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/12.jpg)
Sugar modification Sulphate or Phosphate?
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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
![Page 14: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/14.jpg)
![Page 15: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/15.jpg)
Sulfuric acid
Phosphoric acid
Na
CH3
CH3
Glycan backbone
Glycan backbone
Permethylation
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M+2Na
M+Na
Desulfation: solvolysis with methanolic HCl
Dephosphorylation: HF treatment
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MS2 of PerMe sulfated Lewis A
β-eliminated
Neutral loss of 120
Neutral loss of 120
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Neutral loss of 120
![Page 19: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/19.jpg)
![Page 20: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/20.jpg)
Permethylated sulfo Lewisa is enriched in aqueous phase
Permethylation
Mixture of Lea and sulfo-Lea
Phase partition
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Extraction of PerMe Glycans
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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]+
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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
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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
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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
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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
![Page 27: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/27.jpg)
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
![Page 28: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/28.jpg)
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
![Page 29: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/29.jpg)
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
![Page 30: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/30.jpg)
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
![Page 31: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/31.jpg)
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
![Page 32: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/32.jpg)
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
![Page 33: In-gel O-glycan release In-gel digestion of protein Lecture.pdfKA-O2islet-N-glycan-IT-1402-660_090309144159 #1-5 RT: 0.00-0.16 AV: 5 NL: 6.32 T: ITMS + c NSI Full ms3 1401.70@cid35.00](https://reader033.vdocument.in/reader033/viewer/2022051811/601cdaed21477a00df0ddaa1/html5/thumbnails/33.jpg)
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
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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
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Day 3, Data analysis
Proteomics, method, protein identification (Toshi, Mayumi)
Tools for glycan analysis (MT, MP)
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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
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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
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Chemical release of N-glycan
Anhydrous hydrazine (80oC)
(Hydrazine monohydride 90oC)
Unique sugar sequence, modification
Highly fucosylated N-glycan, etc