Introduction of Proteomic-1

CCRC LIDPEG training course

4/22/2013

Toshihiko Katoh

Gel excision ~ Reduction/alkylation

Protein identification

1. Separation

2. Digestion

3. Peptide analysis

1D, 2D gel separation

(Purification of target protein)

Denaturation (reduction&alkylation)

Enzymatic treatment • Trypsin • Glu-C • Asp-N • chymotrypsin, etc.

Peptide purification (C18 resin)

Mass spectrometry • ESI-MS • MALDI-TOF

Data analysis Protein ID!!

Timeline

(depends)

1 day

1/2 day

1 day (O/N)

1/2 day

1/2 day

1D separation of mixture of protein (You will have a ready-to-excise gel)

M 1 2 3 4 5 6 M 1 2 3 4 5 6

CBB staining Alcian Blue staining (Sulfated and/or sialylated glycans)

Saliva mucin

80ul saliva

1 millimeter cube

Gel excision

Wash and destain the gel

40mM Ambic Acetonitrile (ACN)

Swelling (hydrating)

Shrinking (dehydrating)

Destained gel

Repeat 3 times

Reduction and alkylation (carboxymethylation)

• 10 mM DTT in 40 mM Ambic • 55 mM Iodacetamide (IAA) in 40 mM Ambic

S

S

S

S

S

S

SH

SH

SH

HS

HS

HS

Add 10mM DTT Change to 55mM IAA

At 55°C for 1 h At RT for 1 h In the dark

IAA: ICH2CONH2

SH

SH

SH

HS

HS

HS

H2NOCH2CS

H2NOCH2CS

SCH2CONH2 SCH2CONH2

SCH2CONH2 SCH2CONH2

Reduction of S-S bonds Protection of free SH group

Introduction of Proteomic-2

CCRC LIDPEG training course

4/22/2013

Toshihiko Katoh

Trypsin digestion ~ C18-purification

Trypsin digestion

Protease Cleavage site buffer Temp.

Modified-Trypsin C-terminal of K, R 100mM Ambic (pH7.8) + 10mM CaCl2, 37°C

Glu-C (S. aureus V8) C-terminal of E 100mM Ambic (pH7.8) 25°C

Achromobacter protease I (AP-I)

C-terminal of K 20mM Tris-HCl (pH9.0) 37°C

Endoproteinase Asp-N N-terminal of D N-terminal of D, E

30mM Ambic (pH7.8) + 5mM CaCl2

20mM Sodium borate (pH7.0) +10mM CaCl2

40°C 37°C

Lys-N N-terminal of K 20mM Sodium borate (pH7.8) 45°C

• Sequence Grade Modified Trypsin • Trypsin buffer (100mM ambic/10mM CaCl2)

K K R

K R

K Trypsin

Protein Tryptic peptides

Trypsin digestion

Repeat 2 times

Washing with Ambic/ACN

Dry

Incubate at 37°C O/N

Add trypsin On ice

Swell the gel with trypsin sol.

• 40mM Ambic • Acetonitrile • Trypsin (20ug trypsin in 1mL of trypsin buffer)

Peptide extraction

• 20% acetonitrile/ 5% formic acid • 50% acetonitrile/ 5% formic acid • 80% acetonitrile/ 5% formic acid

20% ACN 50% ACN 80% ACN Trypsin

Speed Vac Plastic tube

Aqueous phase (0.1% Formic acid)

Peptide purification by C18 (octadecylsilane, ODS) resin

C18 (hydrophobic)

Peptide (hydrophobic)

Ions Intact glycans

K R

K

Organic phase (80%ACN/0.1% Formic acid)

C18 (hydrophobic)

Peptide hydrophobic

K R

K

Peptide purification by C18 (octadecylsilane, ODS) resin

• C18 spin column (Microspin, Nest group) • Buffer A: 0.1% Formic acid • Buffer B: 80% acetonitrile/0.1% formic acid

(Wash C18) 300uL of Buffer B, 1,000xg x2

(Sample reconstitution) Add 10uL of Buffer B, mix and 300uL of Buffer A

(Equilibration) 300uL of Buffer A, 1,000xg x3

(Binding) Load the sample, 800xg Put the flow-through back, 800xg 300uL of Buffer A, 800xg x2

C18 resin

(Elution) 250uL of Buffer B, 1,500xg x2

Dry up by Speed Vac

Introduction of Proteomic-3

CCRC LIDPEG training course

4/22/2013

Toshihiko Katoh

LC-MS/MS analysis

(nano)LC-MS/MS analysis (DEMO)

Ion Trap (IT)

Orbitrap (FT)

Spray (ionization)

Separation by C18

Detection

LTQ instrument A B

Repeat cycle over chromatogram

time

Full MS

MS/MS ….

m/z

m/z m/z m/z

Pressure

Sample loader “nitrogen bomb”

Peptide solution