We thank Philamer Calses and Kobe Yuen for critically reading the manuscript. We are thankful to Randy Yen for excellent technical advice.

1. Generation of a Library of Extracellular Human Proteins
<ol>
	<li>Compile a list of cell surface receptors or secreted proteins of interest to build the protein microarray library. Specific protein families (for example, the immunoglobulin superfamily) or proteins selectively expressed in particular cell types can be selected for the study.</li>
	<li>For cell surface receptors, determine the extracellular domain (ECD) boundaries by identifying the signal peptide and transmembrane regions using software tools. Some o...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+combined+transmembrane+topology+and+signal+peptide+prediction--the+Phobius+web+server.">Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server.</a> Nucleic Acids Research. 35 (Web Server issue), W429-W432 (2007).</li><li>Bernsel, A., Viklund, H., Hennerdal, A., Elofsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TOPCONS:+consensus+prediction+of+membrane+protein+topology.">TOPCONS: consensus prediction of membrane protein topology.</a> Nucleic Acids Research. 27 (Web Server issue), W465-W468 (2009).</li><li>Gonzalez, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+the+mammalian+extracellular+proteome+for+regulators+of+embryonic+human+stem+cell+pluripotency.">Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (8), 3552-3557 (2010).</li><li>Durocher, Y., Perret, S., Kamen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+and+high-throughput+recombinant+protein+production+by+transient+transfection+of+suspension-growing+human+293-EBNA1+cells.">High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells.</a> Nucleic Acids Research. 30 (2), E9 (2002).</li><li>Battle, T., Antonsson, B., Feger, G., Besson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+mammalian+protein+expression,+purification,+aliquoting+and+storage+pipeline+to+assemble+a+library+of+the+human+secretome.">A high-throughput mammalian protein expression, purification, aliquoting and storage pipeline to assemble a library of the human secretome.</a> Combinatorial Chemistry &amp; High Throughput Screening. 9 (9), 639-649 (2006).</li><li>Clark, H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secreted+protein+discovery+initiative+(SPDI),+a+large-scale+effort+to+identify+novel+human+secreted+and+transmembrane+proteins:+a+bioinformatics+assessment.">The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment.</a> Genome Research. 13 (10), 2265-2270 (2003).</li></ol>

A significant number of orphan receptors remain in the human genome, and novel interacting partners continue to emerge for extracellular proteins with previously characterized ligands. Defining the receptor-ligand interactions in human and model organisms is essential to understand the mechanisms that dictate cellular communication during homeostasis, as well as dysregulation leading to disease, and therefore inform new or improved therapeutic options. Nevertheless, detection of extracellular protein interactions by wide...

The method reviewed here describes the printing of a collection of extracellular proteins in a microarray format, followed by a method for screening of a target of interest against this library. We have identified protein multimerization as a crucial step for detection of interactions characterized by low binding affinities. To enhance detection of these interactions, we describe a protocol based on multimerization of the query protein of interest using microbeads.
Secreted and cell surface-expressed proteins (collectively termed extracellular proteins) along with their interacting partners are key regulators of cellular communication, signaling and interaction with the microenvironment. They are, therefore, essential in regulating many physiological and pathological processes. Approximately a quarter of the human genome (&#8776;5,000 proteins) encodes for extracellular proteins, which, given their significance and accessibility to systematically delivered drugs, represent key targets for drug development1. Consequently, extracellular proteins represent more than 70% of the protein targets with known pharmacological action for approved drugs on the market, known as the &#34;druggable proteome&#34;. Despite their importance and abundance, the extracellular protein-protein interaction (ePPI) networks remain remarkably underrepresented in the available databases. This is fundamentally due to the complex biochemical nature of the extracellular proteins, which precludes their characterization using most available technologies2. Firstly, membrane proteins are difficult to solubilize, a process that often involves harsh washing conditions and detergents; secondly, extracellular proteins often lack relevant post-translational modifications such as glycosylation that are absent when these proteins are expressed in commonly used heterologous systems. Finally, interactions between receptors, such as co-receptors expressed on immune cells, are often transient and characterized by very low affinities (KD in the ~1 &#956;M to &#62;100 &#956;M range). Altogether, the nature of these proteins and their binding partners render most widely utilized technologies, such as affinity purification/mass spectrometry (AP/MS) or yeast-two-hybrid, unsuitable for detection of interactions in the extracellular space2,3.
In an effort to overcome these technical challenges and accelerate the discovery of novel interactions for extracellular proteins, we have developed a high coverage extracellular protein microarray4,5. Microarrays offer the advantage of generating high-density surfaces with small amounts of sample, and are generally amenable to high throughput studies. Protein microarray-based studies have previously provided relevant insights into protein interactions for several model organisms, albeit mainly focusing on cytosolic interactions or on specific protein families6,7,8. In contrast, limited work has been done to investigate extracellular protein interactions using this technology. We have developed a protein microarray method to enable studies of ePPIs by building a comprehensive and highly diverse library of purified secreted proteins and single transmembrane (STM) receptors expressed as recombinant extracellular domains (ECD) fused to common tags for affinity purification4. The success of the protein microarray screens relies heavily on the establishment of a high quality protein library. For expression of both the library and query protein, mammalian cells or insect cells were preferentially chosen as heterologous expression systems, to ensure proper addition of post-translational modifications such as glycosylation or disulphide bonds. SDS-PAGE, size exclusion chromatography and multi-angle laser light scattering are techniques commonly utilized to assess recombinant protein quality. The protein library is then spotted onto epoxysilane slides and stored at -20 &#176;C for long-term use. Protein concentrations above of 0.4 mg/mL are recommended for the protocol described below. Therefore, low-expressing proteins may require a concentration step prior to sample printing and storage. Nevertheless, a main advantage of this technique is the small volume of protein required (50 &#956;g of protein is sufficient to perform &#62;2,000 screens), alongside minimal query protein consumption (20-25 &#956;g per duplicates screen). Using the protocol and equipment described here, and provided libraries are available, results for individual query proteins can be generated within one working day.
A major challenge in detecting protein interactions in the extracellular environment arises from their characteristically weak or transient nature, which precludes identification by most commonly used methodologies. Increasing the binding avidity greatly improves sensitivity for detection of weak protein interactions9,10,11. Based on this principle we developed a method to multimerize the query proteins (expressed as Fc fusion) using protein A-coated beads4,5. To avoid any potential inactivation of the query protein by random labeling, we instead label an irrelevant human immunoglobulin G with Cy5 and add it along with the query protein to the protein A beads, thus eliminating any artifacts due to the direct conjugation of a dye to the protein of interest. Given the micromolar affinities of several co-receptor pairs, the multivalent complexes greatly enhance signal to noise ratio, compared to Fc-fusion query proteins screened as soluble proteins4.
In summary, the goal of this protocol is to describe the preparation of microarray slides containing a pre-existing extracellular protein library for identification of receptor-ligand interactions. We review the steps for slide printing, followed by a protocol for screening of a protein of interest against the extracellular protein library. Moreover, we describe a method for enhanced detection of ePPIs based on microbeads to achieve increased avidity of the protein under study. The extracellular protein microarray technology described here represents a fast, robust and effective approach for screening and detecting novel ePPI with low false-positive ratios, and by utilizing only microgram quantities of the query protein under investigation. This technology has fueled multiple studies that have provided relevant insights into previously unknown cellular functions and signaling pathways for a variety of receptors12,13, including viral immunoregulators14, and can be utilized to de-orphanize any extracellular protein of interest.

<table>
	<tbody>
		<tr>
			<td>Ultra Pure MB Grade glycerol</td>
			<td>USB Corporation</td>
			<td>56-81-5</td>
			<td>Protein storage</td>
		</tr>
		<tr>
			<td>SeptoMark blocking buffer&#160;</td>
			<td>Zeptosens</td>
			<td>BB1, 90-40</td>
			<td>Blocking buffer microarray slides</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Roche</td>
			<td>03-117-957-001</td>
			<td>Slide control for mask fitting (optional)</td>
		</tr>
		<tr>
			<td>Polypropylene multiwell plates</td>
			<td>Greiner Bio One</td>
			<td>82050-678</td>
			<td>Protein storage</td>
		</tr>
		<tr>
			<td>Polypropylene multiwell plates</td>
			<td>Arrayit</td>
			<td>MMP384</td>
			<td>Slide printing</td>
		</tr>
		<tr>
			<td>NanoPrint LM60, or similar contact microarrayer</td>
			<td>Arrayit</td>
			<td>NanoPrint LM60, or similar contact microarrayer</td>
			<td>Slide printing</td>
		</tr>
		<tr>
			<td>Micro spotting pins</td>
			<td>Arrayit</td>
			<td>Micro spotting pins</td>
			<td>Slide printing</td>
		</tr>
		<tr>
			<td>ZeptoFOG blocking station</td>
			<td>Zeptosens</td>
			<td>ZeptoFOG blocking station, 1210</td>
			<td>Block slides after printing</td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Thermo Fisher</td>
			<td>LP0031</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>Epoxysilane-coated glass slide</td>
			<td>Nextrion Slide E</td>
			<td>1064016</td>
			<td>Microarray slides</td>
		</tr>
		<tr>
			<td>Glass holder and slide rack set</td>
			<td>Wheaton</td>
			<td>900303</td>
			<td>Slide storage</td>
		</tr>
		<tr>
			<td>Cy5 monoreactive dye</td>
			<td>GE Healthcare</td>
			<td>PA23031</td>
			<td>Albumin labeling</td>
		</tr>
		<tr>
			<td>Cy5 monoreactive dye</td>
			<td>GE Healthcare</td>
			<td>PA25001</td>
			<td>Human IgG labeling</td>
		</tr>
		<tr>
			<td>Pro-spin desalting column</td>
			<td>Princeton Separations</td>
			<td>CS-800</td>
			<td>Remove free dye</td>
		</tr>
		<tr>
			<td>Adhesive aluminum foil seal</td>
			<td>AlumaSeal</td>
			<td>F-384-100</td>
			<td>Seal stock plates</td>
		</tr>
		<tr>
			<td>Polypropylene cryogenic vials</td>
			<td>Corning</td>
			<td>430658</td>
			<td>Master vials for protein library storage</td>
		</tr>
		<tr>
			<td>Protein A microbeads</td>
			<td>Miltenyi</td>
			<td>120-000-396</td>
			<td>Query protein multimerization</td>
		</tr>
		<tr>
			<td>Human IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>&#160;009-000-003</td>
			<td>Irrelevant IgG for labeling</td>
		</tr>
		<tr>
			<td>Protein A</td>
			<td>Sigma</td>
			<td>&#160;P7837</td>
			<td>Microarray slide blocking</td>
		</tr>
		<tr>
			<td>Hybridization station, a-Hyb or similar</td>
			<td>Miltenyi</td>
			<td>Hybridization station, a-Hyb or similar</td>
			<td>Automated microarray processing (optional)</td>
		</tr>
		<tr>
			<td>GenePix 4000B scanner or similar</td>
			<td>Molecular Devices</td>
			<td>GenePix 4000B scanner or similar</td>
			<td>Slide scanning</td>
		</tr>
		<tr>
			<td>GenePix Pro or equivalent data extraction software</td>
			<td>Molecular Devices</td>
			<td>GenePix Pro or equivalent data extraction software</td>
			<td>Data processing</td>
		</tr>
		<tr>
			<td>Signal P4.1</td>
			<td>DTU Bioinformatics, Technical University of Denmark</td>
			<td>online software</td>
			<td>Prediction tool to determine presence and location of signal peptide cleavage sites</td>
		</tr>
		<tr>
			<td>TMHMM 2.0 server</td>
			<td>DTU Bioinformatics, Technical University of Denmark</td>
			<td>online software</td>
			<td>Prediction of transmembrane helices in proteins</td>
		</tr>
		<tr>
			<td>Phobius</td>
			<td>Stockholm Bioinformatics Center</td>
			<td>online software</td>
			<td>A combined transmembrane topology and signal peptide predictor</td>
		</tr>
		<tr>
			<td>TOPCONS</td>
			<td>Stockholm University</td>
			<td>online software</td>
			<td>Prediction of membrane topology and signal peptides</td>
		</tr>
	</tbody>
</table>

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.

A schematic of the workflow for the extracellular protein microarray technology is shown in Figure 1. Once the microarray slides containing the extracellular protein library are available, the screening of the protein of interest and data analysis can be completed within one day. Many physiologically relevant interactions between membrane-embedded receptors are characterized by very weak binding strengths (KD in the micromolar range). To improve de...

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

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 ...

This method can help answer key questions in the field of protein protein interactions by enabling the detection of novel binding partners for extracellular proteins, both in high throughput and with high sensitivity. The main advantage of this method is that, with just a few micrograms of protein, you can generate hundreds of slides using the micro array printer. Use a standard micro arrayer for slide printing.This instrument has a capacity of 57 slides per run, uses a print head holding 48 spotting pins, and can accommodate up to 8, 000 spots per slide. Generate working plates using 384 well plates at 10 microliters of sample per well from the stock plates. Perform this step manually, prior to slide printing using the micro arrayer.Then spot proteins with quill type spotting pins onto epoxy silane slides at 60%humidity to prevent dehydration of the protein spots. Cy3 labeled bovine serum albumin can be spotted in duplicate between each protein sample to visualize the array for mask fitting. Subsequent to printing, remove the protein micro array slides from the humidified environment.Next, use an ultrasonic fogger to generate a fine mist of blocking solution that settles onto the slide surface. Then, block the slides overnight with 5%milk in PBST to enactivate the surface. Store slides at minus 20 degrees Celsius in 50%glycerol to prevent freezing.Interactions between extracellular proteins are often characterized by their low affinities. To enable the detection of these interactions, by increasing the binding avidity, we developed a multivalent approach based on the capture of the queried protein expressed as FC tagged extracellular domains on protein A coated micro beads. Spin the carrier IgG, that has been labeled with Cy5 mono reactive dye as described in the text protocol.To calculate the molar ratio of the query protein and Cy5 IgG, divide the molecular weight of the query protein by the molecular weight of the IgG and multiply by 40 micrograms per milliliter to determine the concentration of Cy5 IgG needed. Once all the ratios have been determined form the micro bead protein complex by combining the FC tagged query and the Cy5 IgG with the protein A micro beads. Mix the suspension in PBS on a tube rotator for 30 minutes at room temperature, protected from light.To begin the screening, warm the prepared slides at room temperature before loading onto the hybridization station. To perform the screening, first wash the micro array with PBST for one minute to remove residual glycerol. Load 200 microliters of one milligram per milliliter protein A in PBS 5%milk and incubate for 30 minutes to prevent uncomplexed protein A micro beads from binding to FC tagged proteins that may be present in the micro array.After the hybridization station washes the slides with PBST, load 200 microliters of the query micro bead complex, in the presence of one milligram per milliliter protein A and incubate for 30 minutes. Following hybridization and washing of the slides by the hybridization station, rinse slides with water and place them in individual 50 milliliter conical tubes. Dry the tubes by spinning at 900 times G for five minutes.Finally, scan the slides with a micro array scanner appropriate to detect Cy3 and Cy5 fluorescence by exciting at 532 and 635 nanometers, respectively. Shown here is a representative image of a printed slide. The Cy3 labeled bovine serum albumin spots are in green.The spots were printed in duplicate as a quality control of the printing process. Representative results of an orphan protein, screened for identification of binding partners using the extracellular protein micro array technology, is shown here. The duplicate red spots are identified as a hit for the screened query protein.This method that is described is highly versatile and can be used for the printing of a gamut of protein libraries, be they specific protein families or large compilations of proteins such as ours. Any protein of interest can be evaluated. While attempting this procedure it is important to handle the slides with care and to ensure that they do not dry out so as to prevent the loss of activity of the proteins printed.Following the screen of a protein of interest, it is highly advised to further validate any hits observed by using orthogonal technologies, such as surface plasmon resonance. After its development, this technique has paved the way for researchers to study extracellular protein interactions in humans.

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JoVE Journal

Biochemistry

7.1K 조회수.  Genentech. 이 방법은 높은 처리량과 높은 감도 모두 세포 외 단백질에 대한 새로운 결합 파트너의 검출을 가능하게함으로써 단백질 단백질 상호 작용 분야에서 주요 질문에 대답하는 데 도움이 될 수 있습니다. 이 방법의 주요 장점은 몇 마이크로그램의 단백질만 으로 마이크로 어레이 프린터를 사용하여 수백 개의 슬라이드를 생성할 수 있다는 것입니다. 슬라이드 인쇄에 표준 마이크...