in-gel o-glycan release in-gel digestion of protein...

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 1134.00@cid35.00 660.00@cid35.00 [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 1401.70@cid35.00 660.00@cid25.00 [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