High Throughput Screening of Peptide Substrates for Tyrosine Kinases | Biowire Fall 2011

Featuring PRECISIO® Kinases and PEPscreen™ Custom Peptide Library


 

Biowire Fall 2011 — Screening — microRNA Target Identification

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Danhui Wang, Fan Zhang, Tao Zhao, Fei Zhong, and Keming Song
Sigma® Life Science

Abstract

Protein Tyrosine Kinases (PTKs) play important roles in modulating a wide variety of cellular events, including differentiation, proliferation, metabolism, and apoptosis. Therefore, identification of the substrates for PTKs is vital for basic research and drug development. We report a high-throughput ELISA-based method to identify peptide substrates for class-specific and/or enzyme-specific PTKs. The amino acid sequences of the 13-mer tyrosine peptides for the library were generated using a proprietary algorithm developed for all potential protein substrates of PTKs. Next, the peptides were synthesized using PEPscreen™, a proprietary peptide synthesis platform, and then subjected to a screening of 39 PTKs using an ELISA-based method. The 173 sequences selected were classified into different groups with the reactivity to single, multiple, or all PTKs, and further validated. Validation results indicated that this screening method has a very high sensitivity and reproducibility. The combination of our algorithm for selecting peptide sequences, the PEPscreen peptide synthesis platform, and ELISA-based assay using PRECISIO® Kinases, provides a successful high-throughput system for the screening of PTK peptide substrates.

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Introduction

Kinases are a large group of proteins modulating a wide variety of cellular events, including differentiation, proliferation, metabolism, and apoptosis1. There are 518 protein kinases in the human genome, responsible for phosphorylation of over 30% of all cellular proteins2. Thus, kinases represent an important target for drug discovery3 and a better understanding of kinase pathways and their substrates would greatly impact basic research and drug development.

Kinases mediate their effector functions through phosphorylation of specific amino acid residues in target protein substrates4. Protein kinases are divided into two major classes based on the target amino acid present within the substrates: 1) Protein Serine/Threonine Kinases (PSKs) and 2) Protein Tyrosine Kinases (PTKs). The availability of known substrates for a given kinase represents a potential limiting factor for a kinase assay. Here, we report a high-throughput ELISAbased method to identify peptide substrates for class-specific and/ or enzyme-specific protein kinases. Furthermore, this assay is suitable for detecting kinase activity both in vivo and in vitro. A peptide library containing 13-mer tyrosine peptides was generated using an algorithm developed in-house for all potential protein substrates of protein kinases. The peptides were synthesized using PEPscreen, a proprietary peptide synthesis platform, and then subjected to a screen using an ELISA-based method featuring PRECISIO Kinases.

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Materials and Methods

Unless otherwise indicated, all reagents and materials used in this work were obtained from Sigma. The biotinylated peptides were synthesized on a 96-well format. PTKs were PRECISIO Kinases, obtained from Sigma. Phosphorylated and unphosphorylated tyrosine peptides (Biotin-RRLIEDAEYAARG) were obtained from AnaSpec, Inc. (San Jose, CA). SigmaScreen™ Streptavidin-coated 384-well plate (S8686) was used to anchor biotinylated peptides. Anti-phosphotyrosine monoclonal antibody (P5872), anti-mouse IgG alkaline phosphatase conjugate (A3562), and p-nitrophenyl phosphate liquid substrate system (p-NPP, N7653) were used to detect phosphorylated tyrosine peptides. The optical density was obtained using SpectraMax Plus 384 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA).

Screening Procedure Selection of Tyrosine Peptides
The potential substrates of identified kinases were obtained by searching public databases (Phospho.ELM, and PhosphoSite). The FASTA sequence file including the amino acid sequences of the substrates were compiled and loaded onto KinasePhos database (http://kinasephos.mbc.nctu.edu.tw/index.html) searching for the potential phosphorylation sites of each substrate. The output files were modified by a computer program to identify the 13-mer peptides covering the potential phosphorylation sites. Commercially available peptide substrates for tyrosine kinases were used as positive controls for screening.

Preparation of Peptide Library for Screening
Peptides were reconstituted with 50% acetonitrile at a final concentration of 5 mM. Four 96-well plates of peptides were reformatted into one 384-well plate. The 5 mM peptides were diluted to 0.5 mM with Tris-buffered saline (TBS) as working solutions for screening.

Kinase Reaction
The kinase reaction buffer, kinase, and ATP quantities for each reaction were used as indicated in the product information. 2.5 μl of each 0.5 mM peptide was aliquoted into each well of a 384-well plate followed by adding 22.5 μl of kinase reaction mixture including kinase reaction buffer, ATP, and kinase. The plate was incubated at 30 °C for 15 minutes with constant shaking of 120 rpm. 6 μl of 0.5 M EDTA was then added into each well to stop the kinase reaction.

ELISA Assay
The kinase reaction solutions were transferred into a 384-well Streptavidin-coated plate followed by a shaking incubation of one hour at room temperature. The solutions were removed, followed by three washes with 50 μl of Tris-buffered saline with TWEEN-20 (TBST). 40 μl of mouse anti-phosphotyrosine antibody (1:2,000 dilution) was added into each well followed by a shaking incubation of one hour at room temperature. The solutions were removed, followed by four washes with 50 μl of TBST. 40 μl of anti-mouse IgG conjugated alkaline phosphatase (1:30,000 dilution) was added into each well followed by a shaking incubation of one hour at room temperature. The solutions were removed followed by four washes with 50 μl of TBST. 40 μl of p-NPP was added into each well, followed by a shaking incubation of 30 minutes at room temperature. 8 μl of 3 M sodium hydroxide was added into each well to stop the enzymatic reaction. The optical absorptions at 405 nm were obtained.

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Conclusions

The algorithm developed in house for selecting peptide sequences works well as 50% of the peptides selected showed reactivity to single or multiple kinase. The ELISA-based assay offers both sensitivity and selectivity for the screening of peptide substrates for tyrosine kinases, and PRECISIO Kinases provide suitable activity and consistent results in high-throughput kinase assays.

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Results

With Sigma’s PEPscreen™ Custom Peptide Synthesis, we generated a peptide library with 384 peptides for substrate screening. All peptides were lyophilized in a 96-well plate format. The success rate for peptide synthesis is over 99%. Upon reconstitution of peptides in suitable solution (50% acetonitrile), peptides were subjected to highthroughput ELISA screening for kinase activity with each given protein kinase in 384-well plate. Prior to running the screen, we synthesized a set of biotinylated peptide substrates specific for kinase Abl, Src, IGF1R, or non-specific peptide substrates for most PTKs to test the binding selectivity of SA-coated plate and then tested the antibody specificity for phosphorylated tyrosine peptides by ELISA using an anti-phosphotyrosine antibody. The results (Figure 1) indicate that the ELISA-based assay provides both sensitivity and selectivity for peptide screening with specific detection of phosphorylated tyrosine peptides. Next, the optimal amount of biotinylated peptide for binding to SA-coated plate was identified to be 10 ng in a total of 50 μl reaction (Figure 2A). Also, the optimal dilution of Sigma’s Antiphosphotyrosine monoclonal antibody (P5872) was 1:1000 (Figure 2B). Primary screen was conducted with 384 peptides from the PTK library against each of 39 PTKs using PRECISIO Kinases. The number of positive peptide substrates screened for each PTK was summarized (Table 1). Based on the primary screening results, 170 peptides were selected as potential substrate candidates for single or multiple PTKs and validated by a second round of assays with three replicates for each peptide. Figure 3 shows an example of the validation results for Tyrosine kinases of Src family against selected candidate peptides from primary screening. The validation data are consistent and positively correlate with more than 80% of results obtained in primary screening. Among those validated peptides, some were specific to single kinases; some for groups of non-receptors or receptors, and some for both receptors and non-receptors (Table 2). Finally, seven peptides were selected and tested for their reactivity to each of the 39 PTKs selected (Table 3).

For more information, visit sigma.com/precisiokinases.

Results: Establishment and Optimization of Screening Platform

Elisa-Based Assay Offers Both Sensitivity and Selectivity for the Screening of Peptide Substrates
Figure 1. Elisa-Based Assay Offers Both Sensitivity and Selectivity for the Screening of Peptide Substrates. Nine biotinylated peptides were synthesized and kinase assays were performed with Abl or Src kinase as described in Materials and Methods. The phosphorylated tyrosine residue was detected by an ELISA-based assay using anti-phosphotyrosine antibody. The graph results are the averages of two replicates.
Kemptide: Biotin-LRRASLG-OH (a serine peptide as negative control)
Abltide: Biotin-EAIYAAPFAKKK-OH
SrcPeptide1: Biotin-KVEKIGEGTYGVVYK-OH
IGF1Rtide: Biotin-KKKSPGEYVNIEFG-OH
SrcPeptide2: Biotin-IYGEF-OH
SrcPeptide3: Biotin-TSTEPQYQPGENL-OH
MostPTK1: Biotin-KKKGPWLEEAYGWLDF-OH
MostPTK2: Biotin-RRLIEDAEYAARG-OH

Optimization of ELISA-Based Assay Platform
Figure 2. Optimization of ELISA-Based Assay Platform. A) Four biotinylated peptides were synthesized and then coated on the Streptavidin plate at 5 μg/ml, 1 μg/ml, 0.2 μg/ml, and 40 ng/ml. The ELISA-based assay was performed as described in Materials and Methods. Kemptide: Biotin-LRRASLG-OH; pS-Kemptide: Biotin-LRRApSLG-OH; Y-peptide: Biotin-RRLIEDAEYAARG-NH2; pY-peptide: Biotin-RRLIEDAEpYAARG-NH2. B) 50 μl of pY-peptide (200 ng/ml) was used to coat Streptavidin plate. The phosphorylated tyrosine was detected by a series dilution of anti-phosphotyrosine antibody following the protocol described in Materials and Methods.

Validation of Selected Peptides from Primary Screening
Figure 3. Validation of Selected Peptides from Primary Screening A total of 170 peptide substrates, divided in three groups based on different tyrosine protein kinases, have been selected after primary screening. Validation of peptide candidates was performed by ELISA assay with three repeats for each enzymatic reaction. This figure shows the validation results of activity assay for Tyrosine kinases of Src family against selected candidate peptides. The validation data are consistent and positively correlate more than 80% of results obtained in primary screening.

Table 2. Validation of Selected Peptides from Primary Screening.
  Number of Peptides
Single PTK 47
Non-receptor PTKs 14
Receptor PTKs 17
Non-receptor or Receptor PTKs 96

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Results: Screening of Peptide Substrates for 39 Tyrosine Kinases



Table 1. Primary Screening Results.

Kinase
Number of Peptide Screened Number of Positive Hits Range of Fold / Background Cut-off Value for Selection
AXL 384 0 1-2.3 2.5
EphA1 384 9 1-10.6 2.5
EphA2 384 20 1-11.4 3
EphB1 384 49 1-15.3 4
EphB2 384 48 1-15.3 2.5
EGFR 384 71 1-11.8 4
ERBB2 384 8 1-7.5 2.5
ERBB4 384 13 1-10.7 2.5
FGFR1 384 34 1-10 3
FGFR2 384 55 1-12 4.5
FGFR3 384 5 1-11 4
FGFR4 384 3 1-11.8 2.5
Met 384 12 1-8 3
IR 384 3 1-7.6 2.5
IGFI-R 384 15 1-19.5 3
TRKA 384 18 1-13.2 4
TRKB 384 56 1-9.3 3
PDGFα-R 384 13 1-6.3 3
PDGFβ-R 384 15 1-5.9 3
KIT 384 7 1-4.8 2.5
KDR 384 60 1-10.8 3
FLT3 384 30 1-10.7 3
Abl 384 12 1-12 6
BTK 384 8 1-6.6 2.5
CSK 384 9 1-7.3 3
FAK 384 10 1-5.6 2.5
FES 384 25 1-13 4.5
JAK2 384 62 1-11.6 7
ZAP70 384 1 1-3 2.5
Src 384 28 1-15 7
BLK 384 54 1-12.2 3.5
FGR 384 14 1-5.5 2.5
FYN 384 19 1-15.6 4
HCK 384 35 1-19.9 6.5
LCK 384 13 1-15.9 4
LynA 384 40 1-13.9 3.5
LynB 384 13 1-13.8 3
YES 384 44 1-11.7 4
FRK 384 20 1-10.7 4
Table 3. Reactivity of Selected Peptide Substrates with 39 PTKs. X: Positive Reactivity

Kinase

58

125

174

363

97

102

103
AXL              
EphA1         X X X
EphA2     X   X X X
EphB1     X   X X X
EphB2     X     X X
EGFR     X     X X
ERBB2         X    
ERBB4             X
FGFR1     X       X
FGFR2     X X X X X
FGFR3         X    
FGFR4             X
Met         X    
IR              
IGFI-R     X X     X
TRKA         X   X
TRKB     X X X   X
PDGFα-R         X    
PDGFβ-R         X    
KIT             X
KDR           X X
FLT3           X X
Abl             X
BTK X           X
CSK           X X
FAK         X   X
FES X         X X
JAK2              
ZAP70              
Src X X       X X
BLK X X       X X
FGR         X    
FYN X X       X X
HCK X X       X X
LCK X       X X X
LynA X X       X X
LynB X       X X X
YES X X       X X
FRK         X X X

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Materials

     

References

  1. Hunter, T. Signaling—2000 and beyond. Cell. 2000;100:113–27.
  2. Manning, G, et al. Evolution of protein kinase signaling from yeast to man. Trends in Biochem Sci. 2002;27:514–20.
  3. Noble, M.E., et al. Protein kinase inhibitors: insights into drug design from structure. Science. 2004;303:1800–5.
  4. Hunter, T. A thousand and one protein kinases. Cell. 1987;50:823–9.

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