AF was supported by a starting grant from the European Research Council under the European Community&#39;s Seventh Framework Programme (FP7/2007-2013) / ERC Grant agreement n&#176; 203413 and by the Minerva Center for Bio-hybrid complex systems.HA and AI are supported by the Dalia and Dan Maydan Fellowship for advanced degree students at the Hebrew University of Jerusalem.

1. Designing a Peptide Array
<ol>
	<li>Divide the sequence of the target protein into partly overlapping 10-20 residues peptides. Vary the amount of overlap on the specific experiment and the resources of the performing lab, but in principle the longer the overlap the better. When designing the peptides take into account known secondary structure elements in the protein that can be responsible for the interaction.</li>
	<li>Order the designed peptide array through commercial vendors. Here, use peptides covalently bound to the array through the C-terminus.</li>
</ol>
2. Blocking Non-specific Binding
<ol>
	<li>Make a 50 mM Tris or Phosphate buffer solution containing 0.05% Tween 20 (TBST/PBST) and adjust to the desired pH with a measured amount of HCl or NaOH (in order to know the precise ionic strength) and the desired ionic strength with NaCl. Here, use a pH of 7.5 and an ionic strength of 150 mM.</li>
	<li>Make a blocking solution of 2.5% (w/v) skimmed milk powder in TBST/PBST.</li>
	<li>To prevent non-specific binding, immerse the array in 5 ml of blocking solution. Incubate the array for 2-4 hr at room temperature or overnight at 4 &#186;C on a shaker.</li>
</ol>
3. Incubating with the Protein
<ol>
	<li>Wash the array first with 5 ml of blocking solution for 30 sec and then twice with 5 ml TBST/PBST for 5 min on a shaker at room temperature.</li>
	<li>Incubate the washed array with 5 ml of His-tagged protein solution containing 2.5% (w/v) skimmed milk powder to prevent non-specific binding. Here, use 4.5 &#181;M of protein solution (STIL 500-650) dissolved in the described blocking solution. 
	NOTE: Usually 5-10 &#181;M protein are used for the screening, but the protein concentration can be even as low as 2-3 &#181;M, depending on the binding affinity and the local concentrations of the peptides that bound on the array (efficiency of the synthesis). The buffer in which the protein is dissolved can vary but is common to dilute the protein using the same blocking solution described above.</li>
	<li>Incubate the array in the protein solution for 3-8 hr at room temperature or overnight at 4 &#186;C.</li>
</ol>
4. Incubating with the Antibody
<ol>
	<li>Wash the array three times with 5 ml TBST/PBST. The first wash is for 30 sec followed by two 5 min washes on shaker at room temperature.</li>
	<li>Incubate the washed array with 5 ml of diluted HRP-conjugated antibody (1:1,500) in an incubation buffer that contains the same ingredients at the same concentrations as the blocking solution, for 1 hr on a shaker at room temperature. The antibody can bind either the target protein or the tag.</li>
	<li>Perform control experiments testing the interaction of the antibody with peptides on the array, especially if the array contains peptides from the target protein (e.g., for oligomerization studies) by repeating the same protocol without step 3, incubating with the protein.</li>
	<li>Wash the array three times with 5 ml TBST/PBST. The first wash is for 30 sec followed by two 5 min washes on shaker at room temperature.</li>
</ol>
5. Reading the Array
<ol>
	<li>Carry out chemiluminescence development with an ECL western blotting substrate kit.</li>
	<li>Perform the detection with a luminescent image analyzer.</li>
	<li>Analyze the results. Make sure that both duplicates on the array show the same signals, so the results are reliable. If the structure of the protein partner is known, search on the structure for the binding peptides and look for the binding sites. Even peptides that are far in the sequence can be close together in the tertiary structure and create a binding site.</li>
</ol>

<ol><li>Benyamini, H., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+peptides+to+study+protein%E2%80%93protein+interactions%5BTitle%5D)+AND+%22Future+Medicinal+Chemistry%22%5BJournal%5D)">Using peptides to study protein–protein interactions.</a> Future Medicinal Chemistry. 2 (6), 989-1003 (2010).</li>
<li>Geysen, H. M., Wagner, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isotope+or+mass+encoding+of+combinatorial+libraries%5BTitle%5D)+AND+%22Chemistry+%26+Biology%22%5BJournal%5D)">Isotope or mass encoding of combinatorial libraries.</a> Chemistry & Biology. 3 (8), 679-688 (1996).</li>
<li>Frank, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spot-synthesis%3A+an+easy+technique+for+the+positionally+addressable%2C+parallel+chemical+synthesis+on+a+membrane+support%5BTitle%5D)+AND+%22Tetrahedron%22%5BJournal%5D)">Spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support.</a> Tetrahedron. 48 (42), 9217-9232 (1992).</li>
<li>Katz, C., Levy-Beladev, L., Rotem-Bamberger, S., Rito, T., Rudiger, S. G., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+protein-protein+interactions+using+peptide+arrays%5BTitle%5D)+AND+%22Chem+Soc+Rev%22%5BJournal%5D)">Studying protein-protein interactions using peptide arrays.</a> Chem Soc Rev. 40 (5), 2131-2145 (2011).</li>
<li>Hansen, L. B., Buus, S., Schafer-Nielsen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+Mapping+of+Linear+Antibody+Epitopes+in+Human+Serum+Albumin+Using+High-Density+Peptide+Arrays%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Identification and Mapping of Linear Antibody Epitopes in Human Serum Albumin Using High-Density Peptide Arrays.</a> PLoS ONE. 8 (7), (2013).</li>
<li>Uecker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+substrate+peptide+for+the+FLT3+receptor+tyrosine+kinase%5BTitle%5D)+AND+%22British+Journal+of+Haematology%22%5BJournal%5D)">A substrate peptide for the FLT3 receptor tyrosine kinase.</a> British Journal of Haematology. 144 (1), 127-130 (2009).</li>
<li>Gabizon, R., Faust, O., Benyamini, H., Nir, S., Loyter, A., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure-activity+relationship+studies+using+peptide+arrays%3A+the+example+of+HIV-1+Rev-integrase+interaction%5BTitle%5D)+AND+%22Med.+Chem.+Commun%22%5BJournal%5D)">Structure-activity relationship studies using peptide arrays: the example of HIV-1 Rev-integrase interaction.</a> Med. Chem. Commun. 4 (1), 252-259 (2013).</li>
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<li>Amartely, H., David, A., Lebendiker, M., Benyamini, H., Izraeli, S., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+STIL+protein+contains+intrinsically+disordered+regions+that+mediate+its+protein-protein+interactions%5BTitle%5D)+AND+%22Chemical+Communications%22%5BJournal%5D)">The STIL protein contains intrinsically disordered regions that mediate its protein-protein interactions.</a> Chemical Communications. 50, Cambridge, England. 5245-5247 (2013).</li>
<li>Izraeli, S., Lowe, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+SIL+gene+is+required+for+mouse+embryonic+axial+development+and+left-right+specification%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The SIL gene is required for mouse embryonic axial development and left-right specification.</a> Nature. 399 (6737), 691-694 (1999).</li>
<li>Izraeli, S., Colaizzo-Anas, T., Bertness, V. L., Mani, K., Aplan, P. D., Kirsch, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+the+SIL+gene+is+correlated+with+growth+induction+and+cellular+proliferation%5BTitle%5D)+AND+%22Cell+Growth+Differ%22%5BJournal%5D)">Expression of the SIL gene is correlated with growth induction and cellular proliferation.</a> Cell Growth Differ. 8 (11), 1171-1179 (1997).</li>
<li>Arquint, C., Sonnen, K. F., Stierhof, Y. -D., Nigg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((a+Cell-cycle-regulated+expression+of+STIL+controls+centriole+number+in+human+cells%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">a Cell-cycle-regulated expression of STIL controls centriole number in human cells.</a> Journal of Cell Science. 125 (5), 1342-1352 (2012).</li>
<li>Tang, C. J., Lin, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+human+microcephaly+protein+STIL+interacts+with+CPAP+and+is+required+for+procentriole+formation%5BTitle%5D)+AND+%22Embo+J%22%5BJournal%5D)">The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation.</a> Embo J. 30 (23), 4790-4804 (2011).</li>
<li>Vulprecht, J., David, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((STIL+is+required+for+centriole+duplication+in+human+cells%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">STIL is required for centriole duplication in human cells.</a> Journal of Cell Science. 125 (5), 1353-1362 (2012).</li>
<li>Campaner, S., Kaldis, P., Izraeli, S., Kirsch, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sil+phosphorylation+in+a+Pin1+binding+domain+affects+the+duration+of+the+spindle+checkpoint%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Sil phosphorylation in a Pin1 binding domain affects the duration of the spindle checkpoint.</a> Mol Cell Biol. 25 (15), 6660-6672 (2005).</li>
<li>Kasai, K., Inaguma, S., Yoneyama, A., Yoshikawa, K., Ikeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SCL%2FTAL1+interrupting+locus+derepresses+GLI1+from+the+negative+control+of+suppressor-of-fused+in+pancreatic+cancer+cell%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">SCL/TAL1 interrupting locus derepresses GLI1 from the negative control of suppressor-of-fused in pancreatic cancer cell.</a> Cancer Res. 68 (19), 7723-7729 (2008).</li>
<li>Chaturvedi, P., Sudakin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chfr+regulates+a+mitotic+stress+pathway+through+its+RING-finger+domain+with+ubiquitin+ligase+activity%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Chfr regulates a mitotic stress pathway through its RING-finger domain with ubiquitin ligase activity.</a> Cancer Res. 62 (6), 1797-1801 (2002).</li>
<li>Olaussen, K. a, Commo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synergistic+proapoptotic+effects+of+the+two+tyrosine+kinase+inhibitors+pazopanib+and+lapatinib+on+multiple+carcinoma+cell+lines%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Synergistic proapoptotic effects of the two tyrosine kinase inhibitors pazopanib and lapatinib on multiple carcinoma cell lines.</a> Oncogene. 28 (48), 4249-4260 (2009).</li>
<li>Reingewertz, T. H., Britan-Rosich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+the+Vif%E2%80%93A3G+interaction+using+peptide+arrays%3A+A+basis+for+anti-HIV+lead+peptides%5BTitle%5D)+AND+%22Bioorganic+%26+Medicinal+Chemistry%22%5BJournal%5D)">Mapping the Vif–A3G interaction using peptide arrays: A basis for anti-HIV lead peptides.</a> Bioorganic & Medicinal Chemistry. 21 (12), 3523-3532 (2013).</li>
<li>Katz, C., Benyamini, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+basis+of+the+interaction+between+the+antiapoptotic+Bcl-2+family+proteins+and+the+proapoptotic+protein+ASPP2%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Molecular basis of the interaction between the antiapoptotic Bcl-2 family proteins and the proapoptotic protein ASPP2.</a> Proc Natl Acad Sci U S A. 105 (34), 12277-12282 (2008).</li>
<li>Katz, C., Zaltsman-Amir, Y., Mostizky, Y., Kollet, N., Gross, A., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+Basis+of+the+Interaction+between+Proapoptotic+Truncated+BID+%28tBID%29+Protein+and+Mitochondrial+Carrier+Homologue+2+%28MTCH2%29+Protein%3A+KEY+PLAYERS+IN+MITOCHONDRIAL+DEATH+PATHWAY%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Molecular Basis of the Interaction between Proapoptotic Truncated BID (tBID) Protein and Mitochondrial Carrier Homologue 2 (MTCH2) Protein: KEY PLAYERS IN MITOCHONDRIAL DEATH PATHWAY.</a> Journal of Biological Chemistry. 287 (18), 15016-15023 (2012).</li>
<li>Rosenberg, M. M., Ronen, D., Lahav, N., Nazirov, E., Ravid, S., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+resolution+characterization+of+myosin+IIC+tailpiece+and+its+effect+on+filament+assembly%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">High resolution characterization of myosin IIC tailpiece and its effect on filament assembly.</a> Journal of Biological Chemistry. 288 (14), 9779-9789 (2013).</li>
</ol>

The authors have nothing to disclose.

Peptide array screening is an excellent tool for identifying the binding sites of a target protein in its partners. This assay is fast and easy to perform and the results can be obtained in one or two working days. Peptide array screening is versatile and can be used for many purposes. It can be used for characterizing post translation modifications such as phosphorylation and identifying substrates of kinases. For example: the FLT3 kinase, which is related to acute myeloid leukemia, phosphorylates Tyr-containing substrate peptides found by a peptide array screening6. The inhibitory properties of the tyrosine kinase inhibitors pazopanib and lapatinib was tested using peptide array screening23. Peptides array can be used to identify the sites that mediate protein-protein interactions. Examples of such proteins studied in our lab using this method are: the interaction between the HIV-1 Vif protein and cellular A3G protein was characterized using a peptide array24; peptide array screening was used to study the interactions of the anti-apoptotic BCL-2 family with the pro-apoptotic ASPP2 protein25 and to study the binding of tBID, a member of BCL-2 family, with the mitochondrial protein MTCH226; the intra-molecular interaction between Myosin IIC domains was analyzed using peptide array screening27. Here we present an example of how we used peptide array screening for identifying the sites that mediate the STIL-CHFR interaction, a protein-protein interaction important in proliferation and cancer14. Revealing the exact binding site can serve as basis for the rational design of inhibitors of the studied interaction.
The peptide array screening is not a quantitative assay. It is semi-quantitative since the amount of peptide in each spot on the array varies due to difference in the synthesis yield and peptide purity. This in turn depends on the peptide sequence, length, etc. The variation in yield and purity can also lead sometimes to false negative or false positive results. Comparison between the intensities within the same array can be made, resulting in semi-quantitative ranking. However such comparison between different arrays or different experiments with the same array is not reliable. To quantify the binding, the peptides found in the peptide array screening should be synthesized and tested for binding the target protein using biophysical methods such as fluorescence anisotropy, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), etc. Such assays are not always sensitive to weak binding since they require a high concentration of the protein to detect such binding. The peptide array screening, on the other hand, is very sensitive to weak binding. Thus, some of the interactions observed in the array screening could not be observed or quantified using methods such as fluorescence anisotropy14. The fact that peptide array screening requires very low concentrations of protein combined with the high sensitivity of the assay make the peptide array a useful method for screening protein-peptide interactions in a broad range of affinities.
False negative results can also be due to the fact that linear peptides may not properly represent the 3D structure of the binding site in the protein. This is however not a problem with intrinsically disordered proteins, which are well represented by peptides. False positive results can also be caused by binding of the antibody to the array. These can be detected by performing a control experiment as described in the protocol section 4.3. False negative results can also be obtained if the protein epitope that is recognized by the antibody become unexposed upon binding to the peptide array. This problem is not common but can be solved by using a polyclonal antibody against the protein.
The peptide array screening can also be used for improving lead peptides, including Alanine scan and SAR studies7. Once the binding site of the target is known, an array based on its sequence can be designed with multiple modifications including replacement of each residue to Alanine (Ala scan) to identify the important residues for the interaction. In a similar way, SAR studies can be performed by changing different properties of the binding peptides. These include varying the length of the peptide, replacing residues by the corresponding D-amino acids and other modifications such as methylation, glycosylation and replacement by non-natural amino acids. Such modified peptides, all based on one target sequence, can be synthesized on one array and screened for binding the target protein in one fast and easy experiment, providing a wealth of information for improving a potential inhibitor and understanding the molecular mechanism of the interaction.
The methodology for peptide array screening is highly flexible and changes in the protocol can be applied when required. Other blocking compounds beside milk, such as bovine serum albumin (BSA) or sucrose, can be used. If non-specific binding is observed, higher concentrations of milk/BSA/sucrose should be used. The protein can be incubated with the array in any desired buffer, but it is important to add to the buffer milk / BSA / sucrose or any other blocker that is used in the blocking solution, to prevent unspecific binding. The protein concentration should remain relatively low, and strong binding can be observed even with 3-5 &#181;M protein. The volume of incubation in all steps (blocking, incubation with the protein or antibody and washing) should be enough to fully cover the array. Usually 4-5 ml are enough. A Secondary antibody can be used if the primary antibody is not detectable. In this case steps 6 and 7 should be repeated with dilution and incubation times that fit the specific secondary antibody. The detection procedure depends on the antibody. For horseradish peroxidase (HRP) conjugated antibodies, chemiluminescence is commonly used as described in this protocol. Different alternatives can be used for different antibodies, such as fluorescence or electro-chemiluminescence4. The peptide array described herein is for single use only and cannot be reused.
The protocol presented here demonstrates the wide usefulness of peptide arrays. Using a correctly designed array, binding sites between two proteins can be identified in detail. Once the binding site is well characterized it can be used as a drug target site for inhibiting the relevant protein-protein interaction. Based on the binding peptides found in the array screening, inhibitory small molecules can be designed and screened as drug leads.

Protein-protein interactions mediate most of the processes in the living cell. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions. This is because peptides can be easily synthesized and allow the focusing on a specific interaction site on one hand and on multiple protein targets in a high throughput manner on the other hand1,2. Peptide array screening is a fast, easy to perform method for obtaining a large amount of data about the interactions of a target protein with numerous partners in a short time3. Unlike other biochemical or biophysical methods for detecting and analyzing protein-protein interactions, peptide array screening requires a very low concentration of protein and can detect very weak binding. Peptide arrays can be used for many applications in peptide-protein interactions such as mapping of protein-protein or receptor-ligand interaction sites4, homo- or hetero-oligomerization interfaces, characterizing antibodies epitopes5, studying enzyme activities6 and high throughput structure-activity relationship (SAR) studies7. For an in-depth review about peptide array screening see Katz et al. 4
Several types of peptide arrays currently exist. There are two major synthetic strategies for making peptide arrays: synthesis of the peptides before attaching them to the solid support, or synthesis of peptides directly on the solid support, mainly using the SPOT technique4,8. The peptides are synthesized on the solid support usually by 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry8. Among the common synthetic schemes are peptide attachment through the N terminus (e.g., JPT pepstar arrays9) and peptide attachment through the C terminus (e.g., PEPSCAN pepchip arrays10, JPT pepspot arrays9 and INTAVIS celluspot arrays11)4. The solid support can vary and so does the chemistry of the peptide coupling to it. Cys-terminated peptides can be attached to glass slides via the thiol group12. The N terminus of a peptide can be covalently bound to a hydroxyl group on the cellulose membrane through esterification of the amino acid attached8.
Here we present a detailed protocol for screening peptide arrays as a method for studying protein-protein interactions. The array we used is the Celluspots array, which is a micro-array containing large amount of spots (duplicates of up to 384 spots) on a small cellulose membrane supported by a glass slide. This enables working with low volumes of protein and antibodies and obtaining significant amount of data per single experiment. This array also contains high peptide density that allows detection of low affinity binding. The array was used for mapping the STIL-CHFR interaction, which is highly important for controlling normal cell proliferation13. Uncontrolled interaction between the two proteins can lead to the development of cancer. By mapping this interaction we found the specific binding site and binding residues14. This paves the way for designing rational inhibitors that inhibit this protein-protein interaction.

<table><tbody><tr><td>Peptide array</td><td>INTAVIS Bioanalytical Instruments</td><td></td><td></td></tr><tr><td>His-probe Antibody (H-3) HRP</td><td>Santa Cruz Biotechnology</td><td>sc-8036 HRP</td><td></td></tr><tr><td>EZ-ECL Kit</td><td>Biological industries</td><td>20-500-120</td><td></td></tr><tr><td>Skim Milk</td><td>Becton, Dickinson and Company</td><td>232100</td><td></td></tr><tr><td>LAS-3000 camera&amp;nbsp;</td><td>FUJI film</td><td></td><td></td></tr></tbody></table>

identifying protein-protein interaction sites using peptide arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

STIL is a highly important centrosomal protein. It controls normal cell division and cell proliferation13,15&#8211;19. STIL interacts with several proteins 18,20,21, and most of the interactions occur through its central part, which is an intrinsically disordered region (IDR)14. We designed an array composed of peptides derived from the STIL binding protein CHFR. CHFR is a tumor suppressor that is induced in response to mitotic stress22. Since the structure of the STIL binding domain of CHFR was unknown at the time, we divided the protein domain to 15 residues long peptides with overlap of 7 residues, as described in step 1.1 in the protocol. We performed a peptide array screening for identifying the CHFR-STIL binding sites, as detailed in the above protocol and illustrated in Figure 1. We used an INTAVIS Celluspot array composed of 384 peptides in duplicates, which can provide a huge amount of information as demonstrated in Figure 2. Using this peptide array we found the binding sites for STIL in its binding protein CHFR (Figure 3). Each black spot in the array represents a CHFR derived peptide that bound STIL. Note that each peptide appears twice since it exists in duplicate, which verifies the reliability of the results. The interaction between STIL and CHFR was previously demonstrated at the protein level13,14. The peptide array screening revealed 7 CHFR-derived peptides that bound STIL IDR, residues 500-650 (Figure 3, Amartely et al.14). These results allowed us to map the binding site of STIL on CHFR. Despite the distance of these 7 peptides on CHFR primary sequence they create a well-defined binding site for STIL in CHFR (Figure 4)14.
<img alt="Figure 1" src="/files/ftp_upload/52097/52097fig1highres.jpg" /> 
Figure 1: A scheme of peptide array screening. A- Designing a peptide array that consists of partly overlapping peptides derived from one or more partner proteins, taking into account their secondary structures; B- Incubating with the target protein; C- Incubating with an antibody for detection; D- Detection (can be done by chemiluminescence, fluorescence, etc.); E- Analyzing the results to reveal the protein binding sites. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52097/52097fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52097/52097fig2highres.jpg" /> 
Figure 2: An example of peptide array screening. Detection achieved using chemiluminescence. The slide contains two identical arrays as duplicates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52097/52097fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52097/52097fig3highres.jpg" /> 
Figure 3: Screening a peptide array for binding of STIL IDR to CHFR protein: 4.5 &#181;M His-tagged STIL 500-650 was screened for binding the array. Each black spot indicates binding between the STIL fragment and the CHFR-derived peptide14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52097/52097fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52097/52097fig4highres.jpg" /> 
Figure 4:The STIL binding site in CHFR shown on the structure of the CHFR Cys rich domain (residues 424-661, PDB ID: 2XPO). The seven peptides that bound STIL in the peptide array screening can be divided to three regions colored pink, orange and red, which are distant in the primary sequence but close together in the 3D structure creating one binding site. This full region forms a binding pocket for STIL in the dimer CHFR14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52097/52097fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Identifying Protein-protein Interaction Sites Using Peptide Arrays at JoVE.com

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

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Nanyang Technological University

Research

JoVE Journal

Biology

18.0K Views.  The Hebrew University of Jerusalem. Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Video: Identifying Protein-protein Interaction Sites Using Peptide Arrays

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Biochemistry

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Immunology and Infection

Predicting Amino Acid Preferences of Protein-Protein Binding Interfaces

Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions

Many protein-protein interactions involve the binding of short protein segments to peptide-binding domains. Usually, such interactions require the recognition of linear motifs with variable conservation. The combination of highly conserved and more variable regions in the same ligands often contributes to the multispecificity of binding, a common property of enzymes and cell signaling proteins. Characterization of amino acid preferences of peptide-binding domains is important for the design of mediators of protein-protein interactions (PPIs). Computational methods are an efficient alternative to the often costly and cumbersome experimental techniques, enabling the design of potential mediators that can be later validated in downstream experiments. Here, we described a methodology using the Pepspec application of the Rosetta molecular modeling package to predict the amino acid preferences of peptide-binding domains. This methodology is useful when the structure of the receptor protein and the nature of the peptide ligand are both known or can be inferred. The methodology starts with a well-characterized anchor from the ligand, which is extended by randomly adding amino acid residues. The binding affinity of peptides generated this way is then evaluated by flexible-backbone peptide docking in order to select the peptides with the best predicted binding scores. These peptides are then used to calculate amino acid preferences and to optionally compute a position-weight matrix (PWM) that can be used in further studies. To illustrate the application of this methodology, we used the interaction between subunits of human interferon regulatory factor 5 (IRF5), previously known to be multispecific but globally guided by a short conserved motif called pLxIS. The estimated amino acid preferences were consistent with previous knowledge about the IRF5 binding surface. Positions occupied by phosphorylatable serine residues exhibited a high frequency of aspartate and glutamate, likely because their negatively charged side chains are similar to phosphoserine.

Author Spotlight: A Computational Approach to Decipher Amino Acid Preferences in Multispecific Protein-Protein Interactions

We describe a methodology based on sequence diversification to estimate the amino acid preferences of multispecific binding sites in protein-protein interactions (PPIs). In this strategy, thousands of potential peptide ligands are generated and screened in silico, thus overcoming some limitations of available experimental methods.

Many protein-protein interactions involve the binding of short protein segments to peptide-binding domains. Usually, such interactions require the ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

A Peptide Microarray Technology for Specificity Profiling of Antibodies

Source: Cornett, E. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55912">Analysis of Histone Antibody Specificity with Peptide Microarrays.</a> J. Vis. Exp. (2017).
This video demonstrates a technique that applies peptide microarray technology to profile the specificity of antibodies recognizing post-translational modifications (PTMs) on histone peptides. A microarray slide containing immobilized fluorophore-conjugated histone peptides with known PTM combinations is treated with target PTM-specific antibodies. This process helps identify the antibody&#39;s specificity for the target and its potential cross-reactivity with non-target PTMs.

1. Designing the Array Slide and Source Plate Layout

	Under the &#39;Plan a slide layout&#39; heading on the ArrayNinja interface, click on the link that ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

Protein-protein Interfaces

Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism&#39;s survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a polypeptide chain or string.
Various computational and biochemical methods are used to study protein interfaces. Laboratory methods, such as affinity purification, mass spectrometry, and protein microarrays, can be used to identify new interactions. Co-immunoprecipitation of proteins and yeast two-hybrid screening are widely used to provide evidence on whether two proteins interact in vitro.&#160; Computer programs can predict PPIs based on similar interactions found in other proteins by comparing protein sequences and three-dimensional structures. Other computational approaches, such as phylogenetic profiling, predict PPIs based on the coevolution of binding partners. Additionally, gene fusion analysis is used to predict interaction partners by finding protein pairs that are fused in the genome of other organisms.&#160;
Proteins typically interact with multiple partners either at the same or different times, and they may contain more than one interaction interface.&#160; Many proteins form large complexes that perform specific functions that can only be carried out by the complete complex. In some cases, these protein interactions are regulated; that is, a protein may interact with different partners based on cellular needs. Further computational and statistical analyses sort such interactions into networks, which are curated in online interactome databases. These searchable databases enable users to study specific protein interactions, as well as design drugs that can enhance or disrupt interactions at the interface.

Protein Complexes and Protein-Protein Interactions

Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism&#39;s survival. Most PPIs ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

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