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/Domain Grb2-SH2 Binding to SH2 Binding Peptides SH-PTP2 Binding to SH2 Binding Peptides SmB P3
Modification-Specific
Binding to Peptide Antibody Binding
80.00 20.00

Protein-protein interactions play central roles 70.00

16.00
10.00
Binding to Proline-Rich Peptides

60.00

in almost all cellular responses. Specific 50.00

SpotMatrix
12.00
8.00
40.00 2uM

interaction between peptide motifs and

RCU
2uM

RCU
10uM
8.00 10uM 6.00
30.00 50uM
50uM
2uM

signaling domains such as SH2 and WW

20.00

RCU
4.00 4.00 10uM

Experimental Design. Peptides were synthesized as

10.00

2.00
50uM
Modification-specific antibodies are widely used by
domains is critical for a variety of signaling 0.00 0.00

15- or 20-mers by Sigma-Genosys using their researchers studying signal transduction pathways.

5
2C
1C

3C

4C

5C
1

5
1C

3C

4C

5C
2C

ep

ep

ep

ep

ep
ep

ep

ep

ep

ep

ep
ep

ep

ep

ep
ep

ep

ep

ep
-10.00

ep

H2P

H2P

H2P

H2P

H2P
H2P

H2P

H2P

H2P

H2P

H2P
0.00

H2P

H2P

H2P

H2P
H2P

2P

H2P

2P
H2P

S
S

S
SH

SH
-4.00

S
S

S
However, their affinity and specificity should be carefully
WBP1 LD10 SmB P3 CTD-S5 cdc25 cdc25-noP

S
S
pathways. Many techniques have been PEPscreenSM technology, a high-throughput peptide -2.00

synthesis platform. Peptides contain an N-terminal evaluated. The SpotMatrix SPR platform is well-suited
developed to study protein binding to peptide Peptides
for this application.
biotin and a diethylene glycol linker between the biotin
motifs. These include ELISA, far western,
and first amino acid residues. The average peptide
immuno-precipitation or pull down assays. purity is 73% for 15mer and 61% for 20mer peptides.
Applied Biosystems has developed a new Biotinylated peptides were spotted onto a NeutrAvidin™ Specificity of SH2 Domain Binding. Left, 100nm of WW Domain Binding Specificity. Left, 5 PM WW pTyr-100 Antibody (CST Binding ) to Peptide SpotMatrix

platform that uses Grating-Coupled Surface
Plasmon Resonance (GC-SPR) for kinetic
measurement of molecular interactions
between unlabeled analytes
Affinity Chip using a Cartesian spotter. Analytes, SH2 domain from Grb2 was flowed over the peptide domain of FBP21 Protein was flowed over the peptide
SpotMatrix. Right, 100nM of GST fusion of SH2-PTP2
including full length proteins or domains, were flowed SpotMatrix. Only SmB and P3 peptides give a binding
over the peptide SpotMatrix. Analyte binding to the protein was flowed over the peptide SpotMatrix. The signal with the supershift assay. Both peptides belong to
peptides can be represented as end-point equilibrium maximum binding was observed with peptide spotted at the same subclass (Class III) of WW domain binding
and binding (i.e. average of the last minute of binding signal 50 PM. The optimal binding motif for Grb2 SH2 domain motifs. Right, 300nM of GST-Nedd4 WW domain was
140

120 Peptides

before dissociation begins) as well as the affinity trace. is pYXNX, which is present in all 5 SH2 peptides flowed over the peptide SpotMatrix. Only WBP1 peptide 100
containing

SPR signal (RCU)

biomolecules immobilized on an affinity chip.
phosphotyrosine

80 1

Affinity constants, such as kon (ka), koff (kd) and KD (Keq), containing phosphotyrosine. The optimal binding motif binds to the WW domain in a dose dependent manner 60
3

The optical design of this platform allows were calculated using the Applied Biosystems 8500 for SH2 domain of SH-PTP2 is pY[I/V]X[V/I] is present in with the supershift assay. The binding specificity of both
40

20
9
7
5

11

simultaneous affinity characterization of up to Affinity Chip Analyzer data analysis software. SH2Pep1 and SH2Pep2. Data above agree with this FBP21 and Nedd4 agrees with literature reports (ref: 0
-20
15
13
Nonphosphorylated peptides

400 targets spotted on a Gold Affinity Chip. reported specificity. Peptides named SH2Pep1C, MCB, 2001; 21(22):7617-7628).
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20

SH2Pep2C, SH2Pep3C, SH2Pep4C and SH2Pep5C

We describe this SpotMatrix SPR technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 are control peptides that have the same sequence as
Grb2Pep4C- Grb2Pep4C- Grb2Pep4- Grb2Pep4- Grb2Pep3C- Grb2Pep3C- Grb2Pep3- Grb2Pep3- Grb2Pep2C- Grb2Pep2C- Grb2Pep1C- Grb2Pep1C- Grb2Pep2- Grb2Pep2- Grb2Pep1- Grb2Pep1-

and its application to protein-peptide

1
50uM 50uM 50uM 50uM 50uM 50uM 50uM 50uM 50uM 50uM 50uM 50uM 50uM 50uM 50uM 50uM

Peptide Binding Protein/Domain

2

3
Grb2Pep4C- Grb2Pep4C-
10uM

Grb2Pep4C-
2uM
10uM

Grb2Pep4-
2uM
Grb2Pep4-
10uM

Grb2Pep4-
2uM
Grb2Pep4-
10uM

Grb2Pep4-
2uM
Grb2Pep3C- Grb2Pep3C-
10uM 10uM

Grb2Pep3C- Grb2Pep3C-
2uM 2uM
Grb2Pep3-
10uM

Grb2Pep3-
2uM
Grb2Pep3-
10uM

Grb2Pep3-
2uM
Grb2Pep2C- Grb2Pep2C- Grb2Pep1C- Grb2Pep1C-
10uM 10uM 10uM 10uM

Grb2Pep2C- Grb2Pep2C- Grb2Pep1C- Grb2Pep1C-

2uM 2uM 2uM 2uM
Grb2Pep2-
10uM

Grb2Pep2-
2uM
Grb2Pep2-
10uM

Grb2Pep2-
2uM
Grb2Pep1-
10uM

Grb2Pep1-
2uM
Grb2Pep1-
10uM

Grb2Pep1-
2uM
SH2Pep1, SH2Pep2, SH2Pep3, SH2Pep4 and 100nM GST-Grb2 Binds to Proline-Rich Peptide P3 Designed to
14-3-3- 14-3-3-6- 14-3-3- 14-3-3-
14-3-3- 14-3-3- 14-3-3- 14-3-3- 14-3-3- 14-3-3- 14-3-3- 14-3-3-
Grb2Pep5C- Grb2Pep5C- Grb2Pep5- Grb2Pep5- GST-Grb2protein
GST-Grb2 Proteinbinding
Bindingtotopeptide
PeptideSpotMatrix
SpotMatrix
interactions to illustrate how this technology is SH2Pep5, respectively, but without a phosphate group Bind to WW Domains
4 4_SpThr_50u 4_SpThr_50u 3_FpThr_50u 3_FpThr_50u 2_SpSer_50u 2_SpSer_50u 1_FpSer_50u 1_FpSer_50u
6_SS_50um 50um 5_FS_50um 5_FS_50um 50uM 50uM 50uM 50uM
m m m m m m m m

5
14-3-3- 14-3-3-6- 14-3-3- 14-3-3-
14-3-3- 14-3-3- 14-3-3- 14-3-3- 14-3-3- 14-3-3- 14-3-3- 14-3-3-
4_SpThr_10u 4_SpThr_10u 3_FpThr_10u 3_FpThr_10u 2_SpSer_10u 2_SpSer_10u 1_FpSer_10u 1_FpSer_10u
Grb2Pep5C- Grb2Pep5C- Grb2Pep5- Grb2Pep5- Binding by SH2
6_SS_10um 10um 5_FS_10um 5_FS_10um 10uM 10uM 10uM 10uM

6
14-3-3-
14-3-3-6-2um
14-3-3- 14-3-3-
m
14-3-3-
m
14-3-3-
m
14-3-3-
m
14-3-3-
m
14-3-3-
m
14-3-3-
m
14-3-3-
m
14-3-3- Grb2Pep5C- Grb2Pep5C- Grb2Pep5- Grb2Pep5-
domain of Grb2
GRB2 9.00

6_SS_2um 5_FS_2um 5_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 2uM 2uM

on the Tyrosine residue.

8.00
TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _29- TB4 _29- TB4 _23- TB4 _23- TB4 _17- TB4 _17- TB4 _12- TB4 _12- TB4 _7- TB4 _7- TB4 _1- TB4 _1-

applied to the study of biomolecular

7

Peptide
Assessing Specificity and Affinity of A
2_50uM 2_50uM 1_50uM 1_50uM 43_50uM 43_50uM 36_50uM 36_50uM 31_50uM 31_50uM 26_50uM 26_50uM 21_50uM 21_50uM 15_50uM 15_50uM 7.00
TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _12-26- TB4 _29- TB4 _29- TB4 _23- TB4 _23- TB4 _17- TB4 _17- TB4 _12- TB4 _12- TB4 _7- TB4 _7- TB4 _1- TB4 _1-
8 6.00
2_10uM 2_10uM 1_10uM 1_10uM 43_10uM 43_10uM 36_10uM 36_10uM 31_10uM 31_10uM 26_10uM 26_10uM 21_10uM 21_10uM 15_10uM 15_10uM

SP R signal (RCU)
TB4 12-26- TB4 12-26- TB4 12-26- TB4 12-26- TB4 _29- TB4 _29- TB4 _23- TB4 _23- TB4 _17- TB4 _17- TB4 _12- TB4 _12- TB4 _7- TB4 _7- TB4 _1- TB4 _1-
9
2_2uM 2_2uM 1_2uM 1_2uM 43_2uM 43_2uM 36_2uM 36_2uM 31_2uM 31_2uM 26_2uM 26_2uM 21_2uM 21_2uM 15_2uM 15_2uM
5.00

interactions.
10
Villin C-term
50uM
Villin C-term
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 _12-26-
9_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
20 4.00

Phosphotyrosine Specific Antibody. 40nM of a

SPR signal (RCU)

Villin C-term Villin C-term 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 _12-26- 3.00
11
10uM 10uM 9_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
15 2.00
Villin C-term Villin C-term 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 12-26- Binding by SH3
B B B B B B B B
phosphotyrosine specific antibody was flowed over the
12
2uM 2uM 9_2uM 9_2uM 8_2uM 8_2uM 7_2uM 7_2uM 6_2uM 6_2uM 5_2uM 5_2uM 4_2uM 4_2uM 3_2uM 3_2uM

13
WW_SmB_50 WW_SmB_50 WW_LD10_5 WW_LD10_5
uM uM 0uM 0uM
WW_CTD-
S5_50uM
WW_CTD-
S5_50uM
Flag
Mut_50uM
Flag
Mut_50uM
Flag_50uM Flag_50uM
Bio18E1-
ls_50uM
Bio18E1-
ls_50uM
ACTH
c1_50uM
ACTH
c1_50uM
PKC
inactivator
PKC
inactivator domain of Grb2
GRB2 1.00

SA NA
NA SA NA
SA NA
SA SA NA
NA SA NA
SA NA
SA 14
WW_SmB_10 WW_SmB_10 WW_LD10_1 WW_LD10_1
uM uM 0uM 0uM
WW_CTD-
S5_10uM
WW_CTD-
S5_10uM
Flag
Mut_10uM
Flag
Mut_10uM
Flag_10uM Flag_10uM
Bio18E1-
ls_10uM
Bio18E1-
ls_10uM
ACTH
c1_10uM
ACTH
c1_10uM
50uM
PKC
inactivator
50uM
PKC
inactivator
10 0.00

affinity chip. Left, end-point binding of phosphotyrosine

10uM 10uM -1.00
PKC PKC
WW_SmB_2u WW_SmB_2u WW_LD10_2 WW_LD10_2 WW_CTD- WW_CTD- Bio18E1- Bio18E1- ACTH ACTH
15 Flag Mut_2uM Flag Mut_2uM Flag_2uM Flag_2uM inactivator inactivator
M M uM uM S5_2uM S5_2uM ls_2uM ls_2uM c1_2uM c1_2uM 5
2uM 2uM

_P _R uM

o P uM

_K _R uM

uM
24 uM

pt 10uM
_C 10_1 M

uM

uM
-S uM

M
0u
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

0u
10

10
1

0
5- 10

0
16

10

10
0

_1
0uM 0uM M M M M e_50uM e_50uM 24_50uM 24_50uM noP_50uM noP_50uM 0uM 0uM M M

_1
B_1
Kon Koff KD

_1
II_

e_
3

KI_ I_

e m I_
5_

3_

A
specific antibody to the peptide SpotMatrix. Only those

_R

id
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

IIC
m

PK _P
17 0

W _LD

-n
0uM 0uM M M M M e_10uM e_10uM 24_10uM 24_10uM noP_10uM noP_10uM 0uM 0uM M M

_S
7

TD

A KA

A
A

_R
W

PK PK
25
PK
9

PK P

W
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

A
dc
SpotMatrix SPR
18 PKA_RII_2uM PKA_RII_2uM PKA_RI_2uM PKA_RI_2uM WW_P3_2uM WW_P3_2uM
SH2Pep1 2.53E+04 2.51E-04 9.92E-09

W
11

W
uM uM e_2uM e_2uM 24_2uM 24_2uM noP_2uM noP_2uM uM uM

_c

A
13

W
NeutrAvidinTM Affinity Chip
DEVD-X DEVD-X Grb2Control4 Grb2Control4 Grb2Control3 Grb2Control3 Grb2Control2 Grb2Control2 Grb2Control1 Grb2Control1 -5
-5

15
19 BSA BSA YVAD_50uM YVAD_50uM MBP1 _50uM MBP1 _50uM

peptides containing phosphotyrosine show binding in

W
_50uM _50uM _10uM _10uM _10uM _10uM _10uM _10uM _10uM _10uM
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20
20 BSA BSA YVAD_10uM YVAD_10uM
DEVD-X
_10uM
DEVD-X
_10uM
MBP1 _10uM MBP1 _10uM
Grb2Control4 Grb2Control4 Grb2Control3 Grb2Control3 Grb2Control2 Grb2Control2 Grb2Control1 Grb2Control1
_2uM _2uM _2uM _2uM _2uM _2uM _2uM _2uM SH2Pep2 8.75E+04 1.37E-03 1.57E-08
SH2Pep2 (HPLC
purified) 7.85E+04 1.56E-03 1.99E-08 the SPR assay. Right, representative affinity trace of
Technology SH2Pep3 5.81E+04 1.23E-03 2.12E-08
SH2Pep4 N/A N/A N/A the same experiment shown at left.
SH2Pep5 4.97E+04 1.13E-03 2.27E-08

Cross-talk Between SH3 and WW Domains. 100nM of

Time (Second) GST-Grb2 full length protein was flowed over the peptide
SPR Biosensor Optics Peptide SpotMatrix. Left, schematic diagram of the SpotMatrix. Grb2 protein contains two SH3 domains pThrAntibody(CST) binding to Peptide SpotMatrix
assay. Right, layout of peptides spotted on the affinity separated by one SH2 domain. Left, GST-Grb2 protein
chip. Over 50 different peptides representing a wide not only binds to phosphotyrosine containing peptides,
Kinetics Analysis of SH2 Binding. SH2-binding
Ambient medium range of protein binding motifs were spotted on the but also to the P3 proline-rich peptide, presumably
Surface Plasmon at peptides were spotted on an affinity chip at Titration of
GGGSGSGSGEQPLpTPVTDLG

sample/metal index nA affinity chip. Peptides represent binding motifs for SH2 through its SH3 domain. Right, a detailed data analysis target
interface concentrations ranging from 0.78PM up to 100PM. Left, concentration
domains, 14-3-3 proteins, Actin, WW domains and from the same experiment shown at left. Among all
120

affinity trace of SH2 domain of Grb2 binding to SH2Pep2 100

O TP Detection system cAMP-dependent kinase. Three concentrations (2PM, GSGSGDDPSpYVNVQ

)
SPR signal (RCU
Light Source Peptide is shown. Right, global fit of the affinity traces proline-rich peptides spotted at 10PM, only P3 shows 80
AGGGRRFHpTWPGGAK
10PM, 50PM) for each peptide were spotted in specific binding. SmB peptide, which is very homologous
60
1

for Grb2-SH2 is shown. The kinetic data from the 3

Polarizer Substrate Prism duplicate. to P3 in sequence, shows no binding. The SH3 binding
40 5

PEPscreenSM-synthesized SH2Pep2 peptide and a

7

of index nS 20 9

11

control (HPLC-purified) SH2Pep2 peptide is very similar. motif, [R/K]XXPXXp or PXXPX[R/K], is very homologous 0

15
13

-20

to P3 sequence. Binding of the Grb2 SH3 domain to S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17S18 S19 S20

Kretschmann prism coupling.

Sam68, from which P3 peptide is derived, is supported
This configuration is commonly found
by reports in the literature (J Cell Biochem. 2002;
in available SPR instrumentation.
Proline-rich Peptide 86(1):99-106).

18
Interaction with WW Mapping the Actin- Assessing Specificity and Affinity of A
SPR signal (RCU)

60
16

Phosphothreonine Specific Antibody. 10nM of a

SPR signal (RCU)

14

Cell Window
TAIR
Grating
Pitch a
50
40
30
5
3
1
12

10

6
and SH3 Domains Thymosin E4
phosphothreonine specific antibody was flowed over the
affinity chip. Left, end-point binding of the antibody to
SPR metal film 20 4
1

7
3

the peptide SpotMatrix. Nonphosphorylated peptides

5

with grating 10
7

Interaction
9

11 0
11

(thickness arbitrary) 0 13

Grb2 SH2 PKA

13

and peptides containing phosphoserine show no

-2

Sample Fluid
15

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

-10 S1 S2
Index nA Support Substrate S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17S18 S19S20

(no optical function) binding. However, peptides containing either

Thymosin E4 is a ubiquitous 43 amino acid polypeptide phosphothreonine or phosphotyrosine show significant
WW domain name WW domain binding peptide Motif group Consensus that is an important mediator of cell proliferation, binding. Right, representative affinity traces of the same
Grating-coupled SPR. This WBP1 [Biot]-[DEG]-GTPPPPYTVG I PPxY
migration, and differentiation. It is believed to be one the experiment are shown.
II
configuration is used in the Applied LD10 [Biot]-[DEG]-SGSGAPPTPPPLPPG PPLP
main G-actin sequestering peptides. It is also an anti-
SmB [Biot]-[DEG]-RPPPPGMRRGPPPPGMRPPR III Proline rich sequence with Arginine
Biosystems SpotMatrix SPR 140 16

tumor target due to its important role in angiogenesis.

SPR signal (RCU)

SPR signal (RCU)

120 14
P3 [Biot]-[DEG]-RGRGAAPPPPPVGRGRGVGP III Proline rich sequence with Arginine
platform. 100

80 1
12

10
1
CTD-S5 [Biot]-[DEG]-GSKGGYSPTpSPSGSG IV pSer/pThr-P

The Kretschmann configuration typically used in SPR

60

40

20
9
7
5
3
8

4
9
7
5
3
cdc25

cdc25-noP
[Biot]-[DEG]-SGSGEQPLpTPVTDLG

[Biot]-[DEG]-SGSGEQPLTPVTDLG
IV pSer/pThr-P

I V c o n t r o l Ser/Thr-P 10.00
Binding to Overlapping Thymosin Peptides
Conclusions
11 2 11

instruments depends on a prism to measure SPR angles. -20

0

15
Anti-pTyr Ab
13
0

-2
15
13
14-3-3 Beta
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10S11S12 S13 S14S15 S16S17 S18 S19S20 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 8.00
In this configuration, incident light hits the gold layer on • We describe the application of SpotMatrix SPR
the opposite side of the immobilized biomolecules, posing 6.00
technology for the characterization of either full-
strict requirements for the optical properties of the support 2uM
length proteins or signal transduction domains,
RCU

substrate and the thickness of the gold layer. In the Proline-rich Peptides on the Affinity Chip. Proline-rich 4.00 10uM
50uM

peptide motifs are known to bind to a number of protein such as SH2, 14-3-3, WW and SH3, binding to
grating-coupled configuration, a fine grating on the chip Binding Specificity of Analyte to Peptide 2.00

SpotMatrix. Upper Left, the SH2 domain of Grb2 domains, such as SH3 and WW domains. WW domains over 50 different biotinylated peptides immobilized
surface provides optical coupling and allows imaging of
protein binds specifically to peptides containing can be further subdivided into four groups based on their 0.00
on a NeutrAvidinTM Affinity Chip.
the entire surface at once, enabling simultaneous real- TB4 1-15 TB4 7-21 TB4 12-26 TB4 17-31 TB4 23-36 TB4 29-43

time binding analysis at every spot on the surface. Here, phosphotyrosine. Upper right, protein kinase A particular binding preferences. Above, peptides -2.00 • Signaling domains show a high degree of
incident light hits the gold layer from the top and through catalytic subunit binds specifically to a peptide derived representing all four binding motifs, along with control specificity when binding to this peptide SpotMatrix.
peptide, were spotted onto the affinity chip. Mapping of the Actin-binding Site on Thymosin E.
the biomolecular layer, avoiding the stringent need for an from protein kinase A inhibitor (PKI). Lower left, anti- • In a separate analysis, we used SpotMatrix SPR
Peptide positions within thymosin protein are indicated by
optical quality support substrate and specific gold layer phosphotyrosine antibody binds specifically to all the
their name. 8.7PM of monomeric G-actin was flowed over to interrogate the binding of Thymosin E4 to G-
thickness. The affinity chips are disposable. peptides containing phosphotyrosine but not actin. A scan of overlapping peptides representing
the peptide SpotMatrix. End-point binding of each
nonphosphorylated peptides or phosphoserine or the entire TE4 protein was used to identify the
WBP1 10PM
individual overlapping peptide comprising thymosin is
SPR SpotMatrix Platform phosphothreonine peptides on the affinity chip. Lower actin-binding site, whereas an alanine scan was
GST-ItchA-WW binding to Peptide SpotMatrix
WBP1 50PM
shown. Peptides 12-26 and 17-31 were shown to have
right, protein 14-3-3 Beta binds specifically to peptide used to identify key residues for the interaction.
the highest binding activity to actin.
SpotMatrix SPR Platform containing phosphothreonine and phosphoserine Buffer • We also show the application of this technology
Adhesive Gasket
In
peptide with the cognate flanking sequence. % RCU Signal of Actin Binding to TB4 Sequnce 12-26 (10uM)
Window Anti-GST Ab to assess specificity as well as affinity for capture
Buffer 160
Out 30
agents such as modification-specific antibodies.
GST-ItchA 140
SPR signal (RCU)

• By combining parallel screening with binding

25

20
120
kinetics, this system improves the workflow and
SH2 Domain-Peptide
1
15
3
100
mdegrees

5
10

SpotMatrix on a 1 cm2 area 5 9

7

80 productivity for performing affinity screening and

11

SPR Image high throughput binding characterization studies.

Interaction
0 13
60
15
-5

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

Working with a SpotMatrix SPR Platform. Left, Up to WW Domain of ItchA Binds Specifically to WBP1 For Research Use Only. Not for use in diagnostic procedures.
Peptide Sequence Full protein The Applied Biosystems 8500 Affinity Chip Analyzer has been developed in collaboration with HTS Biosystems
400 targets are spotted onto a 1 cm2 area of the affinity SH2Pep1 [Biot]-[DEG]-GSGSGKPF[pTyr]VNVEF BCR (Y177)
Peptide. 200nM of GST-ItchA WW domain was flowed Determine Essential Residue in Thymosin for Actin Inc.
AB (Design) and Applera are trademarks and Applied Biosystems is a registered trademark of Applera
chip using standard floating-pin spotter. A 40 Pl flow cell SHC (Y317)
over the peptide SpotMatrix. After washing with buffer, Binding. A panel of sequential alanine substitution Corporation or its subsidiaries in the U.S. and/or certain other countries.
SH2Pep2 [Biot]-[DEG]-GSGSGDDPS[pTyr]VNVQ HTS Biosystems is a trademark of HTS Biosystems, Inc.

is then assembled over the SpotMatrix by attachment of a anti-GST antibody was used to “supershift” enhance the mutants of the peptide 12-26 were used to determine the NeutrAvidin is a trademark of Pierce Biotechnology Inc.
SH2Pep3 [Biot]-[DEG]-GSGLPVPE[pTyr]INQSV ErbB1(Y1068) PEPscreen is a service mark of Sigma-Aldrich Corporation.

self-adhesive window containing a fluid inlet and outlet binding signal. Left, end-point signal of peptide essential residues for actin binding. All peptides were Information is subject to change without notice.
SH2Pep4 [Biot]-[DEG]-GSGVGNPE[pTyr]LNTVQ ErbB2(Y1139)
(center). The assembled affinity chip is then inserted into SpotMatrix binding to ItchA is shown. Of over 50 different spotted at 10PM concentration. The bar graph above WW domain fusion proteins were kindly provided by Dr. Mark T. Bedford, The University of Texas M.D.
SH2Pep5 [Biot]-[DEG]-DTFLPVPE[pTyr]INQSV ErbB1(Y1068) Anderson Cancer Center.
the instrument to measure the binding of an analyte to peptides, including some proline-rich peptides, only depicting the average end-point binding at equilibrium ”Copyright 2004 Applied Biosystems. All rights reserved.
each of the immobilized targets using a CCD camera for Phosphotyrosine containing peptides in the SpotMatrix WBP1 peptide shows significant binding. Right, affinity shows that the Lysine at position 18 (substituted with
imaging the entire surface in real-time (right). trace of the binding to all proline-rich peptides is shown alanine in 12-26-3 mutant) of thymosin is essential for
with the supershift assay. actin binding.