Beads & Chips

Guru Reddy

New SELDI ProteinChip Technology

EC-SELDI-MS Platform

Expression Difference Mapping TM (EDM)

Expression Profiling (Current application)

High Speed Purification (New)

Applications in Clinical, Research & Process Proteomics

IP-SELDI-MS Platform

Interaction Difference Mapping TM (IDM)

Kinase assays and Kinase Protein Interactor Discovery (New)

Protein Interactions & interactor expression difference (New)

Applications in Clinical & Research Proteomics

EC-SELDI-MS Platform

EC-SELDI-MS:

Expression optimization and predictive

purification of recombinant proteins

Steve Cleverley - European Senior Scientist

Lee Lomas – Ciphergen R&D

EC-SELDI-MS Platform to Monitor Protein Expression in High Throughput Fashion Validation of EC-SELDI-MS as a method of recombinant Protein

Detection

Expression Optimization of In vitro Translation Protein Expression Systems

Design of Optimal Purification Strategies for both Tagged and Non-Tagged Proteins

Detection of RTS Expressed His-tagged Proteins using EC-SELDI-MS

Background

RTS (Rapid Translation System™) is an in vitro protein synthesis system

The expression levels of RTS expressed proteins varies considerably.

Currently, expression is monitored by 1D SDS PAGE followed by either Colliodal Blue staining, or Western Blot analysis using anti-His mAbs.

Aim

Use EC-SELDI-MS to capture and detect a range of different RTS expressed His-tagged proteins

Rapid Translation System is the trademark of Roche Diagnostics

EC-SELDI-MS: Expression Monitoring of His-Tagged Proteins

20l Sample incubated with Ni-IDA Cellulose

(Individual column or 96-well HT format)

Wash with Imidazole Buffers

Retentate Chromatography on ProteinChip Arrays

Analyze by SELDI-MS

SAX2 H50

WCX2 NP20

0

1

2

3

4

15000 20000 25000 30000 35000 Da

5000 10000 15000 20000 25000 30000

5000 10000 15000 20000 25000 30000

K-Ras old 10ul

B-Wash 2

K-Ras old 10ul(2)

B-Wash 2(2)

50001000015000200002500030000

EC-SELDI-MS Detects Peaks That Cannot be Detected by SELDI-MS

EC-SELDI-MS

IMAC40-NiCaptureSELDI-MS

00.511.5200002100022000230002400025000

The data above and opposite compares the direct capture of his-tagged K-Ras with on an IMAC40-Ni ProteinChip Array with the capture of K-Ras onto a Ni2+-Resin, followed by profiling of the elutant on a SAX2 ProteinChip® Array. The figure opposite shows that there is a 10-fold increase in sensitivity as a result of the EC-SELDI-MS approach.

The data above and opposite compares the direct capture of his-tagged K-Ras with on an IMAC40-Ni ProteinChip Array with the capture of K-Ras onto a Ni2+-Resin, followed by profiling of the elutant on a SAX2 ProteinChip® Array. The figure opposite shows that there is a 10-fold increase in sensitivity as a result of the EC-SELDI-MS approach.

Molecular Mass (M/z)

Sign

al I

nten

sity

-0.10

0.10.20.3

10000 20000 30000 40000

31407.7+HGFP-Luc

00.25

0.50.75

10000 20000 30000 40000 50000

32227.4+HGST

02.5

57.510

10000 20000 30000 40000 50000

38608.6+H

GFH/pTH-His

0246

20000 30000 40000 50000 60000

46847.8+HMBP

012345

10000 20000 30000 40000 50000 60000

23aPSP (undetected)

0246

10000 20000 30000 40000 50000

CATS (undetected)

05

10152025

10000 20000 30000 40000

28057.5+HGFP

EC-SELDI-MS: Detection of Expressed Proteins in RTS

Could not be detected byColloidal staining & Western Blotting

Detected by Colloidal stainingbut Western can not confirm

0.1hr

1.0hr

1.5hr

2hr

3hr

4hr

8hr

1

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5

28119.5+H

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5

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Molecular Mass (M/z)

Sign

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12

14

0 0.2 0.4 0.6

Final Concentration of Amino Acids(mM)

% S

igna

l Int

ensi

ty

LysateFormula 1

Sign

al I

nten

sity

LysateFormula 2

LysateFormula 2+ AA Mix

EC-SELDI-MS: Expression Optimization of In Vitro Protein Translation System

10

20

30

10

20

30

10

20

30

0

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20

30

10000 20000 30000

26.1kDa

Purification Strategies for tagged Proteins

(eg His, Myc, GST, MBD)

Molecular Mass (M/z)Si

gnal

Int

ensi

ty

FlowThrough

10mMImidazole

100mMImidazole

500mMImidazole

His-eIF4e

Biosepra IDA-celluloseLoaded with Ni2+

Elution of Contaminants100mM Imidazole

Elution of His-eIF4e500mM Imidazole

EC-SELDI-MS: Design of Purification Strategies for Tagged Proteins

26.1kDa 0mM NaCl

50mM NaCl

200mM NaCl

400mM NaCl

15000 20000 25000 30000 35000

0

10

0

10

0

10

0

10

0

10 1M NaCl

Molecular Mass (M/z)

15000 20000 25000 30000 35000

pH9.5

pH8.5

pH7.5

pH6.5

26.1kDa

0

10

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10

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10

Sign

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nten

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Sign

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Strong Anionic ProteinChip Array (SAX)

EC-SELDI-MS: Design of Purification Strategies for Non-Tagged Proteins

100mM Buffer pH6.5 (Binding)

Elution of ContaminantspH6.5, 200mM NaCl

Elution of eIF4e at pH6.5 and 400mM NaCl

His-eIF4e

Biosepra IDA-celluloseLoaded with Ni2+

Elution of Contaminants100mM Imidazole

Elution of His-eIF4e500mM Imidazole

EC-SELDI-MS: Predictive Protein Chromatography™

A Rationale for Scale up Procedures

EC-SELDI-MS is Turning Months into DaysPurification Scouting & Optimization

45000 50000

pH 4.5

pH 4.6

pH 4.7

pH 4.8

pH 5.0

Target moleculeTarget molecule

Binding pH optimizationpH 4.5 – 4.7

Target moleculeTarget molecule

20000 40000 60000

2.5 mS

5.0 mS

7.5 mS

10 mS

Binding I.S. Optimization 7.5 - 10 mS/cm

Elution NaCl conc.

150 mM.

20000 40000 60000 80000

Target moleculeTarget molecule

75 mM NaCl

90 mM NaCl

100 mM NaCl

125 mM NaCl

150 mM NaCl

200 mM NaCl

EC-SELDI-MS: Protein Expression Profiling

OrExpression Difference Mapping ™

Eric Fung, Enrique Dalmasso & BDC

Prostate Cancer Biomarker DiscoveryExpression Difference Mapping

2500 5000 7500 10000 12500

2500 5000 7500 10000 12500

Ret Map 1-4

Ret Map 1-3

Ret Map 1-4(2)

Ret Map 1-3(2)

comp1Difference map

Patient

Control

GelViewTM

2 5 0 0 5 0 0 0 7 5 0 0 1 0 0 0 0 1 2 5 0 0

2 5 0 0 5 0 0 0 7 5 0 0 1 0 0 0 0 1 2 5 0 0

Ret Map 1-4

Ret Map 1-3

Ret Map 1-4(2)

Ret Map 1-3(2)

comp1

2500 5000 7500 10000 12500

0

2

4

639

97.8

6

4467

.6+H

4843

.6+H

6226

.76

6419

.11

6605

.27

7700

.05 79

07.1

180

92.4

+H

8711

.41

8906

9227

.03

9781

.51

9975

.5

2500 5000 7500 10000 12500

0

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3028

.2+H

3994

.89

4469

.89

6229

.73

6422

.12

6615

.94

7516

.78

7699

.22

7912

.93

8098

.46

8908

.66

9234

.28

9404

.62

2500 5000 7500 10000 12500-2.5

0

2.5

5

7.5

Strong anion exchange resin

Fx1 Fx2 Fx3 Fx4

Cu(II) (IMAC3)

Sample+ Urea/CHAPS/TrisHCl pH 9

EC-SELDI-MS: Protein Profiling of Serum

Flow-through

Hepes pH 7.0 eluant

100 mM NaAcetate pH 5 eluant

iPrOH/ACN/TFA

eluant

100 mM NaCitrate pH 3 eluant

Weak cation exchange (WCX2)Low stringency

Fx5 Fx6

100 mM NaAcetate pH 4 eluant

Weak cation exchange (WCX2)High stringency

EC-SELDI-MS increases peak count7500 8000 8500 9000 9500

7500 8000 8500 9000 9500

Total serum

Q Fraction 1

Q Fraction 2

Q Fraction 3

Q Fraction 4

Q Fraction 5

Q Fraction 6

EC-SELDI-MS increases peak count (high mass)

20000 22500 25000 27500 30000

20000 22500 25000 27500 30000

Total serum

Q Fraction 1

Q Fraction 2

Q Fraction 3

Q Fraction 4

Q Fraction 5

Q Fraction 6

40000 45000 50000 55000 60000

40000 45000 50000 55000 60000

Total serum

Q Fraction 1

Q Fraction 2

Q Fraction 3

Q Fraction 4

Q Fraction 5

Q Fraction 6

60000 70000 80000 90000 100000

60000 70000 80000 90000 100000

Total serum

Q Fraction 1

Q Fraction 2

Q Fraction 3

Q Fraction 4

Q Fraction 5

Q Fraction 6

Reproducibility of fractionation4000 6000 8000 10000

4000 6000 8000 10000

Fraction 1

Fraction 2

Fraction 3

Fraction 4

Fraction 5

Fraction 6

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510

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0255075

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Fraction 1

Fraction 2

Fraction 3

Fraction 4

Fraction 5

Fraction 6

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0246

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25000 50000 75000

IP-SELDI-MS: Interaction Difference Mapping ™

Kinase Assays

Protein-Protein Interactions

Zhang Wang, Vaneetha Thulasiraman, Lee Lomas & Tai Tung

Yip Ciphergen R&D

Assay Development: Automated Kinase Assay Protein kinases play a key role in nearly all signal

transduction pathways in cells.

Currently kinase activity is monitored by radioactive or fluorescent methods.

We developed an automatable IP-SELDI-MS method for discovery of new protein interactions and monitoring kinase activity

IP-SELDI-MS: Automated Kinase Assay

Kinase Plate

Filtration orCentrifugation

Collection plate (kinase reaction mix)

Membranefilter

IMAC-Ga(III)ProteinChip array

Sample plate(substrate+/-Drugs, etc.)

Y

Y

Y

Anti-kinase

Kinase

SubstrateProduct

PP

P

ATP

YY

Capture of Phosphopeptide using IMAC-Ga ProteinChip® Arrays

IMAC ProteinChip® Array

IMAC-Ga ProteinChip® Array

Analyze by SELDI-MS

IMAC-Ga Surface Preparation (1.5 hr)

Phospho-peptide Enrichment (1.5 hr)

1400 1500 1600

PS

IP-SELDI-MS: PKCa Activity Titration

0

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0.001 0.01 0.1 1 10

Ave

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PKC (ng/ul)

CVp=25.0%, df=27

1300 1400 1500 1600

1387.9+H1467.8+H1540.0+H

SP

I

IP-SELDI-MS: PKCa Inhibition Curve

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Ave

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PKC Inhibitor (uM)

IC50= 0.3 uM

CVp=17.7%, df=23

Multiplexed Kinase Assays

Kinase 2

Substrate-1

Product-1

PPP

Kinase 1

Substrate-2

Product-2

0204060110012001300140015001046.4+H1244.3+H1469.6+H

Multiplexing of four Kinases (Immunoprecipitation)

PKC

MAPK

AKT

024100001025010500107501100010339.2+H10419.3+H10497.9+H10567.4+HJNK11

1040010600108001100030003050310031503200

11001200130014001500

Multiplexing of Seven Kinases (in soln)

PKC1467.6+1PMAPK

1047.2+1P

AKT1243.6+1P

17001725175017751800

PKA1096.2+1P

SRC1749.6+1P

GSK33029.1+1P

3109.1+2P

3189.1+3P

JNK11

10493.7+2P

10574.4+3P

GSK3b activity in the absence ofAKT GSK3b activity in the presence ofAKT

3029.1+1P

3109.1+2P 3189.1

+3P

A

E

DB C

Advantage of Kinase Assays by IP-SELDI-MS or Interaction Difference Mapping™

Radioactive yes no no

Multiplexing no yes yes

Phospho-site mapping no yes no

P32 SELDI FP

Labeled substrate no no yes

Experimental Goals and Design:

Use purified SH2-GST fusion proteins as targets for protein-protein and protein-peptide interaction

Capture phosphorylated proteins from whole cell lysates of “resting” and “activated” cell lines

GST only was used as a negative control

IP-SELDI-MS: Protein-Protein Interactions

SH2 Domain: A Key Player in Signal Transduction Pathways

SH2 Domains:

Present in many proteins participating in signal transduction pathways

Play a role in intracellular protein-protein interactions via SH2 domain binding

Binding requires a phosphate group, usually at Tyrosine

Near neighbor amino acids provide SH2 domain specificity

SH2 proteins frequently include catalytic and/or SH3 domains

PS2 ProteinChip® Array

SH2-GST Surface

Purified SH2-GST fusion protein

Cell Lysates - Mammalian Cell Lines

SH2-P-Tyr complex

Couple Target SH2 to the Chip

PBS II ProteinChip® Reader

Bind specific SH2-binding Proteins

Wash

Detect Bound Proteins

IP-SELDI-MS: Capture of SH2 Domain Binding Proteins from Whole Cell Lysates:

GST

SH2

crk SH2-Domain Binding Proteins Captured from Activated and Resting Cell Lysates - PS2 ProteinChip

Array

The red, purple traces represent activated cells, the blue, green traces, resting cells

crk-GST fusion protein and GST only were covalently attached to PS2 ProteinChips

20000 40000 60000 80000 100000

35124.841291.382120.736580.168383.829410.6+Hcrk-GSTmonomer

GSTcrk-GST

dimer

crk binding proteinactivation specific

crk bindingprotein(s)

The red, purple traces represent activated cells, the blue, green traces, resting cells

gap-GST fusion protein and GST only were covalently attached to PS2 ProteinChips

Whole cell lysates in 1% Triton X-100, 10% glycerol were diluted 10-fold in TBST. Final protein concentration = 2.0 mg/ml

40000 60000 80000 100000

31387.140336.760366.480326.236732.940339.951966.771229.180327.2

gap SH2-Domain Binding Proteins Captured from Activated and Resting Cell Lysates - PS2 ProteinChip

Arraygap-GSTmonomer

GST

gap bindingprotein(s)

gap-GSTdimergap binding

protein(s)

Zheng Wang, Lee Lomas

Dissociation of human cardiac Troponin complex

Human cardiac Troponin complex:

accessory protein components of the thin filaments in skeletal and cardiac muscles. Three polypeptides-Tn C, I and T (named for their Calcium-bindig, Inhibitory and Tropomyosin-binding activities).

TnC: 18416.623 Da, pI=4.0

TnI: 24007.587 Da, pI=10.0

TnT: 34590.311 Da, pI=5.1

Purpose of the project:

based on binding affinity and other properties, explore conditions to dissociate sequentially the three components in the complex on ProteinChip® Arrays

Introduction

General Protocol

1. Bind antibody on PS10 chips

2. Bind human heart extract (1ul) mixed in 4ul of PBS or other buffers where indicated, 1.5 hr at RT,

3. Wash 3 times with 5 ul of different buffers

4. Transfer 2 ul of the wash to SAX2 or WCX2, which are pre-equilibrated with the same buffer for 5 min

5. Binding on SAX2 or WCX2 for 15 min, rinse all chips with 1mM HEPES, pH 7 in bulk

6. Analyze by SELDI-MS

Although PS10 chips are used here, for low affinity antibodiesUse of antibodies coupled to beads (IP-SELDI-MS) is useful

Capture of Tn C, I and T on PS10

Anti-TnI, 8I7

Mouse IgG

TnC TnTTnI

0

2

4

6

20000 30000 40000

15787.7+H18697.8+H

24196.6+H34405.4+H

0

2

4

6

20000 30000 40000

15833.7+H

18655.3+H 24225.3+H 34469.0+H

Effect of pH on the stability of Tn Complex

0

2.5

5

7.5

10

20000 30000 40000

18678.1+H

23950.9+H

32609.7+H34366.3+H

0

2.5

5

7.5

10

20000 30000 40000

18694.9+H

24176.1+H34375.4+H

TnC TnTTnI

BSA

pH 5

pH 7

Conclusion: low pH increases stability, N=3 experiments

0

2.5

5

7.5

10

20000 25000 30000 35000 40000

18731.4+H

23940.0+H34436.2+H

0

2.5

5

7.5

10

20000 25000 30000 35000 40000

18654.4+H 23957.0+H 34315.5+H

Effect of pH 10 on Tn C

TnC TnTTnI

Anti-TnI, 8I7

Mouse IgG

Conclusion: pH10 and EDTA eliminates TnC binding to the complex

Binding in a high Ca buffer increases TnI intensity

0

5

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15

20000 30000 40000

18410.0+H

24127.7+H

34397.6+H

0

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5

7.5

10

20000 30000 40000

18532.7+H

24128.6+H

34307.1+H

TnC TnTTnI

N=2 experiments

Effect of EDTA on Tn C, I and T interaction

0

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7

TnC TnI TnT

Inte

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2mM EDTA

10mM EDTA

Conclusion: EDTA decreases TnC binding to the complex

Conclusions

Tn C is a calcium-binding protein, so use of high EDTA can decrease or eliminate binding of Tn C from the complex.

The interaction also seems to be charge-dependent, use of high pH can eliminate binding of Tn C from the complex.

TnT-I binding is stronger, so far, only 8M Urea can strip off TnT at the cost of reduced TnI binding to the antibody.

Other conditions have been tried, low concentration of Urea, low % of detergents and salt concentration did not seem to facilitate dissociation.

The complex is significantly stabilized when washing with low pH buffer, a condition maybe desirable for increasing interactions.

Beads & Chip Sales Pitch for Different Market Segments

Process Proteomics Use chromtagraphic beads and/or ProteinChip Arrays (SELDI-MS)

employing retentate mapping to develop purifications protocols then scale up by using the BioSepra beads: EC-SELDI-MS or SELDI-MS

Predictive Protein Chromatography ™ Days instead of months

Clinical Proteomics Use chromatography beads to pre-fractionate the serum before

applying to ProteinChip Arrays for final retentate mapping: EC-SELDI-MS

Expression Difference Mapping ™ More peaks & better reproducibility

Research Proteomics bead-based enrichment/capture step prior to on-chip retentate

mapping, the discovery & detection of many proteins and interactions are dramatically increased: IP-SELDI-MS

Interaction Difference Mapping ™