Binding & Kinetic Analysis of Protein Interactions Probed by Label-Free SpotMatrix SPR Technology in a Peptide Chip Format

Yong Xue, Kathryn Ferrell, Oscar Monera+, Rick Baggio, Yu-Xin Yan, Dina Wassaf, Maura Barbisin, Richard Noble, Michelle Palmer, Jaime Arenas, Stacey Hoge+, Melissa A. Gee +Sigma-Genosys, 1442 Lake Front Circle, Woodlands, TX 77380; Applied Biosystems, 35 Wiggins Avenue, Bedford, MA 01730

Introduction
Protein-protein interactions play central roles in almost all cellular responses. Specific interaction between peptide motifs and signaling domains such as SH2 and WW domains is critical for a variety of signaling pathways. Many techniques have been developed to study protein binding to peptide motifs. These include ELISA, far western, immuno-precipitation or pull down assays. Applied Biosystems has developed a new platform that uses Grating-Coupled Surface Plasmon Resonance (GC-SPR) for kinetic measurement of molecular interactions between unlabeled analytes and biomolecules immobilized on an affinity chip. The optical design of this platform allows simultaneous affinity characterization of up to 400 targets spotted on a Gold Affinity Chip. We describe this SpotMatrix SPR technology and its application to protein-peptide interactions to illustrate how this technology is applied to the study of biomolecular interactions.

Protein/Domain Binding to Peptide SpotMatrix
Experimental Design. Peptides were synthesized as 15- or 20-mers by Sigma-Genosys using their PEPscreenSM technology, a high-throughput peptide synthesis platform. Peptides contain an N-terminal biotin and a diethylene glycol linker between the biotin and first amino acid residues. The average peptide purity is 73% for 15mer and 61% for 20mer peptides. Biotinylated peptides were spotted onto a NeutrAvidin™ Affinity Chip using a Cartesian spotter. Analytes, including full length proteins or domains, were flowed over the peptide SpotMatrix. Analyte binding to the peptides can be represented as end-point equilibrium binding (i.e. average of the last minute of binding signal before dissociation begins) as well as the affinity trace. Affinity constants, such as kon (ka), koff (kd) and KD (Keq), were calculated using the Applied Biosystems 8500 Affinity Chip Analyzer data analysis software.

Grb2-SH2 Binding to SH2 Binding Peptides
80.00 70.00 16.00 60.00 50.00 40.00 30.00 20.00 4.00 10.00 0.00
1 2 H2P ep 2C S H2P ep 3 SH 2P ep 3C S H2P ep 4 S H2P ep 4C S H2P ep 5 SH 2P ep 5C 1C H2P ep H2P ep

SH-PTP2 Binding to SH2 Binding Peptides
20.00

SmB

P3
Binding to Proline-Rich Peptides
10.00

Modification-Specific Antibody Binding
2uM 10uM 50uM

12.00 2uM 10uM 50uM 2uM 10uM 50uM RCU

8.00

RCU

RCU

8.00

6.00

4.00

0.00
1 2 3 4 1C 2C 3C 4C 5 H2P ep H2P ep H2P ep H2P ep H2P ep H2P ep H2P ep H2P ep H2P ep H2P ep H2P ep 5C

2.00

-10.00
S

S

S

S

S

S

S

-4.00

S

S

S

S

0.00 WBP1 -2.00 LD10 SmB P3 CTD-S5 cdc25 cdc25-noP

Peptides

Modification-specific antibodies are widely used by researchers studying signal transduction pathways. However, their affinity and specificity should be carefully evaluated. The SpotMatrix SPR platform is well-suited for this application.

1 1

2

3 Grb2Pep450uM Grb2Pep410uM Grb2Pep42uM 14-3-35_FS_50um 14-3-35_FS_10um 14-3-35_FS_2um

4 Grb2Pep450uM Grb2Pep410uM Grb2Pep42uM

5

6

7 Grb2Pep350uM Grb2Pep310uM Grb2Pep32uM

8 Grb2Pep350uM Grb2Pep310uM Grb2Pep32uM

9

10

11

12

13 Grb2Pep250uM Grb2Pep210uM Grb2Pep22uM

14 Grb2Pep250uM Grb2Pep210uM Grb2Pep22uM

15 Grb2Pep150uM Grb2Pep110uM Grb2Pep12uM Grb2Pep550uM Grb2Pep510uM Grb2Pep52uM TB4 _115_50uM TB4 _115_10uM TB4 _115_2uM

16 Grb2Pep150uM Grb2Pep110uM Grb2Pep12uM Grb2Pep550uM Grb2Pep510uM Grb2Pep52uM TB4 _115_50uM TB4 _115_10uM TB4 _115_2uM

Grb2Pep4C- Grb2Pep4C50uM 50uM Grb2Pep4C- Grb2Pep4C10uM 10uM Grb2Pep4C2uM 14-3-36_SS_50um 14-3-36_SS_10um 14-3-36_SS_2um Grb2Pep42uM 14-3-3-650um 14-3-3-610um 14-3-3-6-2um

Grb2Pep3C- Grb2Pep3C50uM 50uM Grb2Pep3C- Grb2Pep3C10uM 10uM Grb2Pep3C- Grb2Pep3C2uM 2uM

Grb2Pep2C- Grb2Pep2C- Grb2Pep1C- Grb2Pep1C50uM 50uM 50uM 50uM Grb2Pep2C- Grb2Pep2C- Grb2Pep1C- Grb2Pep1C10uM 10uM 10uM 10uM Grb2Pep2C- Grb2Pep2C- Grb2Pep1C- Grb2Pep1C2uM 2uM 2uM 2uM

2

Peptide Binding Protein/Domain

3

4

5

14-3-314-3-314-3-314-3-314-3-314-3-314-3-314-3-314-3-3Grb2Pep5C- Grb2Pep5C4_SpThr_50u 4_SpThr_50u 3_FpThr_50u 3_FpThr_50u 2_SpSer_50u 2_SpSer_50u 1_FpSer_50u 1_FpSer_50u 5_FS_50um 50uM 50uM m m m m m m m m 14-3-314-3-314-3-314-3-314-3-314-3-314-3-314-3-3Grb2Pep5C- Grb2Pep5C14-3-34_SpThr_10u 4_SpThr_10u 3_FpThr_10u 3_FpThr_10u 2_SpSer_10u 2_SpSer_10u 1_FpSer_10u 1_FpSer_10u 10uM 10uM 5_FS_10um m m m m m m m m 14-3-314-3-314-3-314-3-314-3-314-3-314-3-314-3-314-3-3Grb2Pep5C- Grb2Pep5C5_FS_2um 4_SpThr_2um 4_SpThr_2um 3_FpThr_2um 3_FpThr_2um 2_SpSer_2um 2_SpSer_2um 1_FpSer_2um 1_FpSer_2um 2uM 2uM TB4 _2943_50uM TB4 _2943_10uM TB4 _2943_2uM TB4 _2943_50uM TB4 _2943_10uM TB4 _2943_2uM TB4 _2336_50uM TB4 _2336_10uM TB4 _2336_2uM TB4 _2336_50uM TB4 _2336_10uM TB4 _2336_2uM TB4 _1731_50uM TB4 _1731_10uM TB4 _1731_2uM TB4 _1731_50uM TB4 _1731_10uM TB4 _1731_2uM TB4 _1226_50uM TB4 _1226_10uM TB4 _1226_2uM TB4 _1226_50uM TB4 _1226_10uM TB4 _1226_2uM TB4 _721_50uM TB4 _721_10uM TB4 _721_2uM TB4 _721_50uM TB4 _721_10uM TB4 _721_2uM

6

Peptide

7

TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-262_50uM 2_50uM 1_50uM 1_50uM TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-262_10uM 2_10uM 1_10uM 1_10uM TB4 12-262_2uM Villin C-term 50uM Villin C-term 10uM Villin C-term 2uM TB4 12-262_2uM Villin C-term 50uM Villin C-term 10uM Villin C-term 2uM TB4 12-261_2uM TB4 12-261_2uM

8

Specificity of SH2 Domain Binding. Left, 100nm of SH2 domain from Grb2 was flowed over the peptide SpotMatrix. Right, 100nM of GST fusion of SH2-PTP2 protein was flowed over the peptide SpotMatrix. The maximum binding was observed with peptide spotted at 50 M. The optimal binding motif for Grb2 SH2 domain is pYXNX, which is present in all 5 SH2 peptides containing phosphotyrosine. The optimal binding motif for SH2 domain of SH-PTP2 is pY[I/V]X[V/I] is present in SH2Pep1 and SH2Pep2. Data above agree with this reported specificity. Peptides named SH2Pep1C, SH2Pep2C, SH2Pep3C, SH2Pep4C and SH2Pep5C are control peptides that have the same sequence as SH2Pep1, SH2Pep2, SH2Pep3, SH2Pep4 and SH2Pep5, respectively, but without a phosphate group on the Tyrosine residue.

S

S

S

WW Domain Binding Specificity. Left, 5 M WW domain of FBP21 Protein was flowed over the peptide SpotMatrix. Only SmB and P3 peptides give a binding signal with the supershift assay. Both peptides belong to the same subclass (Class III) of WW domain binding motifs. Right, 300nM of GST-Nedd4 WW domain was flowed over the peptide SpotMatrix. Only WBP1 peptide binds to the WW domain in a dose dependent manner with the supershift assay. The binding specificity of both FBP21 and Nedd4 agrees with literature reports (ref: MCB, 2001; 21(22):7617-7628).

pTyr-100 Antibody (CST Binding ) to Peptide SpotMatrix

140 120 SPR signal (RCU) 100 80 60 40 20 0
15 13 9 11 Nonphosphorylated peptides 7 3 5 1 Peptides containing phosphotyrosine

-20

S1 S2

S3

S4 S5

S6

S7

S8

S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20

GST-Grb2 Protein Binding to Peptide SpotMatrix GST-Grb2protein binding to peptide SpotMatrix
Binding by SH2 domain of GRB2 Grb2

100nM GST-Grb2 Binds to Proline-Rich Peptide P3 Designed to Bind to WW Domains
9.00 8.00 7.00 6.00 SP R signal (RCU) 5.00 4.00 3.00 2.00 1.00 0.00 -1.00

9

10

TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-269_50uM 9_50uM 8_50uM 8_50uM 7_50uM 7_50uM 6_50uM 6_50uM 5_50uM 5_50uM 4_50uM 4_50uM 3_50uM 3_50uM TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-269_10uM 9_10uM 8_10uM 8_10uM 7_10uM 7_10uM 6_10uM 6_10uM 5_10uM 5_10uM 4_10uM 4_10uM 3_10uM 3_10uM TB4 12-269_2uM TB4 12-269_2uM TB4 12-268_2uM WW_CTDS5_50uM WW_CTDS5_10uM WW_CTDS5_2uM TB4 12-268_2uM WW_CTDS5_50uM WW_CTDS5_10uM TB4 12-267_2uM Flag Mut_50uM Flag Mut_10uM TB4 12-267_2uM Flag Mut_50uM Flag Mut_10uM TB4 12-266_2uM Flag_50uM TB4 12-266_2uM Flag_50uM TB4 12-265_2uM Bio18E1ls_50uM Bio18E1ls_10uM Bio18E1ls_2uM TB4 12-265_2uM Bio18E1ls_50uM Bio18E1ls_10uM Bio18E1ls_2uM TB4 12-264_2uM ACTH c1_50uM ACTH c1_10uM ACTH c1_2uM TB4 12-264_2uM ACTH c1_50uM ACTH c1_10uM ACTH c1_2uM TB4 12-263_2uM PKC inactivator 50uM PKC inactivator 10uM PKC inactivator 2uM TB4 12-263_2uM PKC inactivator 50uM PKC inactivator 10uM PKC inactivator 2uM

20

11

15

B

B

B

B

B

B

B

B

12

13

SA SA SA SA NA NA NA NA

NA NA NA NA SA SA SA SA

WW_SmB_50 WW_SmB_50 WW_LD10_5 WW_LD10_5 uM uM 0uM 0uM WW_SmB_10 WW_SmB_10 WW_LD10_1 WW_LD10_1 uM uM 0uM 0uM WW_SmB_2u WW_SmB_2u WW_LD10_2 WW_LD10_2 M M uM uM

Binding by SH3 domain of Grb2 GRB2

10

14

Flag_10uM

Flag_10uM

15

16

SpotMatrix SPR Technology
SPR Biosensor Optics
Surface Plasmon at sample/metal interface
P

13

11

18

PKA_RIICA_2 PKA_RIICA_2 PKA_Kemptid PKA_Kemptid PKA_PKI_5- PKA_PKI_5- WW_cdc25- WW_cdc25- WW_cdc25_2 WW_cdc25_2 PKA_RII_2uM PKA_RII_2uM PKA_RI_2uM PKA_RI_2uM WW_P3_2uM WW_P3_2uM uM uM e_2uM e_2uM 24_2uM 24_2uM noP_2uM noP_2uM uM uM BSA BSA YVAD_50uM YVAD_50uM DEVD-X _50uM DEVD-X _10uM DEVD-X _50uM DEVD-X _10uM MBP1 _50uM MBP1 _50uM Grb2Control4 Grb2Control4 Grb2Control3 Grb2Control3 Grb2Control2 Grb2Control2 Grb2Control1 Grb2Control1 _10uM _10uM _10uM _10uM _10uM _10uM _10uM _10uM Grb2Control4 Grb2Control4 Grb2Control3 Grb2Control3 Grb2Control2 Grb2Control2 Grb2Control1 Grb2Control1 _2uM _2uM _2uM _2uM _2uM _2uM _2uM _2uM

NeutrAvidinTM Affinity Chip

19

20

BSA

BSA

YVAD_10uM YVAD_10uM

MBP1 _10uM MBP1 _10uM

SH2Pep2 SH2Pep2 (HPLC purified) SH2Pep3 SH2Pep4 SH2Pep5

8.75E+04 7.85E+04 5.81E+04 N/A 4.97E+04

1.37E-03 1.56E-03 1.23E-03 N/A 1.13E-03

1.57E-08 1.99E-08 2.12E-08 N/A 2.27E-08

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20

Ambient medium index nA Detection system

SPR signal (RCU

Light Source Polarizer Substrate Prism of index nS

Peptide SpotMatrix. Left, schematic diagram of the assay. Right, layout of peptides spotted on the affinity chip. Over 50 different peptides representing a wide range of protein binding motifs were spotted on the affinity chip. Peptides represent binding motifs for SH2 domains, 14-3-3 proteins, Actin, WW domains and cAMP-dependent kinase. Three concentrations (2 M, 10 M, 50 M) for each peptide were spotted in duplicate.

Time (Second)

Kinetics Analysis of SH2 Binding. SH2-binding peptides were spotted on an affinity chip at concentrations ranging from 0.78 M up to 100 M. Left, affinity trace of SH2 domain of Grb2 binding to SH2Pep2 Peptide is shown. Right, global fit of the affinity traces for Grb2-SH2 is shown. The kinetic data from the PEPscreenSM-synthesized SH2Pep2 peptide and a control (HPLC-purified) SH2Pep2 peptide is very similar.

Kretschmann prism coupling. This configuration is commonly found in available SPR instrumentation.

18

SPR signal (RCU)

16 14 12 10 8 6 4 2 0 11 -2 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 13 9 7 5 3

AIR

SPR signal (RCU)

60 50 40
1

Cell Window

Grating Pitch a SPR metal film with grating

30
5

3

Proline-rich Peptide Interaction with WW and SH3 Domains
1

Cross-talk Between SH3 and WW Domains. 100nM of GST-Grb2 full length protein was flowed over the peptide SpotMatrix. Grb2 protein contains two SH3 domains separated by one SH2 domain. Left, GST-Grb2 protein not only binds to phosphotyrosine containing peptides, but also to the P3 proline-rich peptide, presumably through its SH3 domain. Right, a detailed data analysis from the same experiment shown at left. Among all proline-rich peptides spotted at 10 M, only P3 shows specific binding. SmB peptide, which is very homologous to P3 in sequence, shows no binding. The SH3 binding motif, [R/K]XXPXXp or PXXPX[R/K], is very homologous to P3 sequence. Binding of the Grb2 SH3 domain to Sam68, from which P3 peptide is derived, is supported by reports in the literature (J Cell Biochem. 2002; 86(1):99-106).

15

-5 -5

W W

SH2Pep1

2.53E+04

2.51E-04

9.92E-09

W W

9

_S

7

17

PKA_RIICA_1 PKA_RIICA_1 PKA_RII_10u PKA_RII_10u PKA_RI_10u PKA_RI_10u PKA_Kemptid PKA_Kemptid PKA_PKI_5- PKA_PKI_5- WW_cdc25- WW_cdc25- WW_cdc25_1 WW_cdc25_1 WW_P3_10u WW_P3_10u 0uM 0uM M M M M e_10uM e_10uM 24_10uM 24_10uM noP_10uM noP_10uM 0uM 0uM M M

0

5

3

PKA_RIICA_5 PKA_RIICA_5 PKA_RII_50u PKA_RII_50u PKA_RI_50u PKA_RI_50u PKA_Kemptid PKA_Kemptid PKA_PKI_5- PKA_PKI_5- WW_cdc25- WW_cdc25- WW_cdc25_5 WW_cdc25_5 WW_P3_50u WW_P3_50u 0uM 0uM M M M M e_50uM e_50uM 24_50uM 24_50uM noP_50uM noP_50uM 0uM 0uM M M

Kon

Koff

KD

m W B_1 W W _LD 0u W _C 10_1 M TD 0 -S uM 5_ 10 PK uM A _R II_ PK P 10 A KA _P _R uM KI_ I_ 5- 10 _c 24 uM dc _1 25 0 -n o P uM _1 W 0u W M PK _P 3_ A _R 10 IIC uM A _1 PK PK 0 A A _K _R uM e m I_ pt 10uM id e_ 10 uM

WW_CTDFlag Mut_2uM Flag Mut_2uM S5_2uM

Flag_2uM

Flag_2uM

5 1

Assessing Specificity and Affinity of A Phosphotyrosine Specific Antibody. 40nM of a phosphotyrosine specific antibody was flowed over the affinity chip. Left, end-point binding of phosphotyrosine specific antibody to the peptide SpotMatrix. Only those peptides containing phosphotyrosine show binding in the SPR assay. Right, representative affinity trace of the same experiment shown at left.

SPR signal (RCU)

pThrAntibody(CST) binding to Peptide SpotMatrix

GGGSGSGSGEQPLpTPVTDLG

120

Titration of target concentration

100

GSGSGDDPSpYVNVQ
80

)

AGGGRRFHpTWPGGAK
60 3 40 7 20 11 0 15 -20 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17S18 S19 S20 13 9 5 1

20 10 0
15 13 9 11

7

Sample Fluid Index nA

Support Substrate
(no optical function)

-10 S1 S2

S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17S18 S19S20

15

(thickness arbitrary)

Grb2 SH2

PKA

Mapping the ActinThymosin 4 Interaction
Thymosin 4 is a ubiquitous 43 amino acid polypeptide that is an important mediator of cell proliferation, migration, and differentiation. It is believed to be one the main G-actin sequestering peptides. It is also an antitumor target due to its important role in angiogenesis.
Binding to Overlapping Thymosin Peptides 10.00

WW domain name

WW domain binding peptide
[Biot]-[DEG]-GTPPPPYTVG [Biot]-[DEG]-SGSGAPPTPPPLPPG [Biot]-[DEG]-RPPPPGMRRGPPPPGMRPPR [Biot]-[DEG]-RGRGAAPPPPPVGRGRGVGP [Biot]-[DEG]-GSKGGYSPTpSPSGSG [Biot]-[DEG]-SGSGEQPLpTPVTDLG [Biot]-[DEG]-SGSGEQPLTPVTDLG

Motif group
I II III III IV IV PPxY PPLP

Consensus

SPR signal (RCU)

SPR signal (RCU)

Grating-coupled SPR. This configuration is used in the Applied Biosystems SpotMatrix SPR platform. The Kretschmann configuration typically used in SPR instruments depends on a prism to measure SPR angles. In this configuration, incident light hits the gold layer on the opposite side of the immobilized biomolecules, posing strict requirements for the optical properties of the support substrate and the thickness of the gold layer. In the grating-coupled configuration, a fine grating on the chip surface provides optical coupling and allows imaging of the entire surface at once, enabling simultaneous realtime binding analysis at every spot on the surface. Here, incident light hits the gold layer from the top and through the biomolecular layer, avoiding the stringent need for an optical quality support substrate and specific gold layer thickness. The affinity chips are disposable.

WBP1 LD10
140 16

Assessing Specificity and Affinity of A Phosphothreonine Specific Antibody. 10nM of a phosphothreonine specific antibody was flowed over the affinity chip. Left, end-point binding of the antibody to the peptide SpotMatrix. Nonphosphorylated peptides and peptides containing phosphoserine show no binding. However, peptides containing either phosphothreonine or phosphotyrosine show significant binding. Right, representative affinity traces of the same experiment are shown.

SmB P3 CTD-S5
1 3 5 7

Proline rich sequence with Arginine Proline rich sequence with Arginine pSer/pThr-P pSer/pThr-P

120

14 12 10 8 6 4 9 2 0 15 -2 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 13 11

100

80 3 60 5 40 9 20 11 0 15 -20 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10S11S12 S13 S14S15 S16S17 S18 S19S20 7

1

cdc25 cdc25-noP

I V c o n t r o l Ser/Thr-P

Conclusions
• We describe the application of SpotMatrix SPR technology for the characterization of either fulllength proteins or signal transduction domains, such as SH2, 14-3-3, WW and SH3, binding to over 50 different biotinylated peptides immobilized on a NeutrAvidinTM Affinity Chip. • Signaling domains show a high degree of specificity when binding to this peptide SpotMatrix. • In a separate analysis, we used SpotMatrix SPR to interrogate the binding of Thymosin 4 to Gactin. A scan of overlapping peptides representing the entire T 4 protein was used to identify the actin-binding site, whereas an alanine scan was used to identify key residues for the interaction. • We also show the application of this technology to assess specificity as well as affinity for capture agents such as modification-specific antibodies. • By combining parallel screening with binding kinetics, this system improves the workflow and productivity for performing affinity screening and high throughput binding characterization studies.

Anti-pTyr Ab
13

14-3-3 Beta
8.00

6.00 2uM 10uM 50uM
RCU

SPR SpotMatrix Platform

SpotMatrix SPR Platform
Adhesive Gasket Window

In Out

Binding Specificity of Analyte to Peptide SpotMatrix. Upper Left, the SH2 domain of Grb2 protein binds specifically to peptides containing phosphotyrosine. Upper right, protein kinase A catalytic subunit binds specifically to a peptide derived from protein kinase A inhibitor (PKI). Lower left, antiphosphotyrosine antibody binds specifically to all the peptides containing phosphotyrosine but not nonphosphorylated peptides or phosphoserine or phosphothreonine peptides on the affinity chip. Lower right, protein 14-3-3 Beta binds specifically to peptide containing phosphothreonine and phosphoserine peptide with the cognate flanking sequence.

Proline-rich Peptides on the Affinity Chip. Proline-rich peptide motifs are known to bind to a number of protein domains, such as SH3 and WW domains. WW domains can be further subdivided into four groups based on their particular binding preferences. Above, peptides representing all four binding motifs, along with control peptide, were spotted onto the affinity chip.

4.00

2.00

0.00 TB4 1-15 -2.00 TB4 7-21 TB4 12-26 TB4 17-31 TB4 23-36 TB4 29-43

WBP1 10 M
GST-ItchA-WW binding to Peptide SpotMatrix

WBP1 50 M

Mapping of the Actin-binding Site on Thymosin . Peptide positions within thymosin protein are indicated by their name. 8.7 M of monomeric G-actin was flowed over the peptide SpotMatrix. End-point binding of each individual overlapping peptide comprising thymosin is shown. Peptides 12-26 and 17-31 were shown to have the highest binding activity to actin.
% RCU Signal of Actin Binding to TB4 Sequnce 12-26 (10uM)

Buffer Anti-GST Ab Buffer
30

160 140 120

SPR signal (RCU)

25

GST-ItchA
1

20

SpotMatrix on a 1 cm2 area SPR Image

SH2 Domain-Peptide Interaction
Peptide SH2Pep1 SH2Pep2 SH2Pep3 SH2Pep4 SH2Pep5 Sequence [Biot]-[DEG]-GSGSGKPF[pTyr]VNVEF [Biot]-[DEG]-GSGSGDDPS[pTyr]VNVQ [Biot]-[DEG]-GSGLPVPE[pTyr]INQSV [Biot]-[DEG]-GSGVGNPE[pTyr]LNTVQ [Biot]-[DEG]-DTFLPVPE[pTyr]INQSV Full protein BCR (Y177) SHC (Y317) ErbB1(Y1068) ErbB2(Y1139) ErbB1(Y1068)

15 3 5 7 5 11 0 15 -5 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 13 9

10

mdegrees

100 80 60 40 20 0 12-26 12-26-1 12-26-2 12-26-3 12-26-4 12-26-5 12-26-6 12-26-7 12-26-8 12-26-9

Acknowledgements
For Research Use Only. Not for use in diagnostic procedures. The Applied Biosystems 8500 Affinity Chip Analyzer has been developed in collaboration with HTS Biosystems Inc. AB (Design) and Applera are trademarks and Applied Biosystems is a registered trademark of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. HTS Biosystems is a trademark of HTS Biosystems, Inc. NeutrAvidin is a trademark of Pierce Biotechnology Inc. PEPscreen is a service mark of Sigma-Aldrich Corporation. Information is subject to change without notice. WW domain fusion proteins were kindly provided by Dr. Mark T. Bedford, The University of Texas M.D. Anderson Cancer Center. Copyright 2004 Applied Biosystems. All rights reserved.

Working with a SpotMatrix SPR Platform. Left, Up to 400 targets are spotted onto a 1 cm2 area of the affinity chip using standard floating-pin spotter. A 40 l flow cell is then assembled over the SpotMatrix by attachment of a self-adhesive window containing a fluid inlet and outlet (center). The assembled affinity chip is then inserted into the instrument to measure the binding of an analyte to each of the immobilized targets using a CCD camera for imaging the entire surface in real-time (right).

Phosphotyrosine containing peptides in the SpotMatrix

WW Domain of ItchA Binds Specifically to WBP1 Peptide. 200nM of GST-ItchA WW domain was flowed over the peptide SpotMatrix. After washing with buffer, anti-GST antibody was used to “supershift” enhance the binding signal. Left, end-point signal of peptide SpotMatrix binding to ItchA is shown. Of over 50 different peptides, including some proline-rich peptides, only WBP1 peptide shows significant binding. Right, affinity trace of the binding to all proline-rich peptides is shown with the supershift assay.

Determine Essential Residue in Thymosin for Actin Binding. A panel of sequential alanine substitution mutants of the peptide 12-26 were used to determine the essential residues for actin binding. All peptides were spotted at 10 M concentration. The bar graph above depicting the average end-point binding at equilibrium shows that the Lysine at position 18 (substituted with alanine in 12-26-3 mutant) of thymosin is essential for actin binding.