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 of the relevant tools, freely available online, are referenced in the Table of Materials15,16,17,18.</li>
	<li>Synthesize the ECD for the genes of interest and clone into the relevant vector. Secreted proteins or the ECD of STM receptors fused to a number of common affinity tags can be purified from the conditioned media of cells transfected with the appropriate vector, or from baculovirus-insect cell expression systems. 
	Note: Mammalian cell-based systems (such as HEK/293 or CHO cells) are recommended to maximize the likelihood of proper protein folding and addition of relevant posttranslational modifications such as glycosylation.</li>
	<li>Purify the proteins by standard affinity purification methods. Previous efforts have described automated or semi-automated procedures suitable for purification of hundreds of proteins, which can be scaled to generate the set of proteins of interest9,19,20,21. 
	Note: SDS-PAGE, size exclusion chromatography (S), or multi-angle laser light scattering (MALLS) are recommended to assess any non-covalent aggregation and to control for the overall quality of the protein preparations.</li>
	<li>Adjust proteins to 200 or 400 &#956;g/mL whenever possible, and dilute stocks with 80% glycerol for long-term storage in cryogenic vials at -20 &#176;C. These represent master stocks and should be accessed only when necessary.</li>
	<li>Transfer aliquots of each protein (100 &#956;L) to 96-well plates (stock plates), seal with adhesive foil, and store at -20 &#176;C until microarray slide preparation. Stock plates are generated to minimize freeze-thaw cycles and ensure protein stability.</li>
</ol>
2. Extracellular Protein Microarray Printing
<ol>
	<li>Use a standard microarrayer for slide printing. The instrument utilized for the protocol described here has a capacity of 57 slides/run and uses a printhead holding 48 spotting pins. These pins generate spots of ~100 &#956;m diameter separated by a spot-to-spot distance of ~350 &#956;m and draws 0.25 &#956;L per load. Under this configuration, up to 8,000 spots per slide can be accommodated.</li>
	<li>Generate working plates (384 well plates, 10 &#956;L sample/well) from the stock plates. Perform this step manually prior to slide printing using the microarrayer. Then, spot proteins with quill-type spotting pins onto epoxysilane slides at 60% humidity (to prevent dehydration of the protein spots). 
	Note: Cy3-labeled Bovine serum albumin (BSA) can be spotted in duplicates between each protein sample (5 &#956;g/mL in PBS/40% glycerol) to visualize the array for mask fitting (see section 4). Although recommended, this step is optional.</li>
	<li>Subsequent to printing, remove the protein microarray slides from the humidified environment and block them overnight with 5% milk in PBST to inactivate the surface. 
	Note: The preferred approach is to use an ultrasonic fogger to generate a fine mist of blocking solution that settles onto the slide surface.</li>
	<li>Store slides at -20 &#176;C in 50% glycerol to prevent freezing.</li>
</ol>
3. Preparation of Multivalent Bait Complexes
Note: Interactions between extracellular proteins are often characterized by low affinities. To enable detection of these interactions by increasing binding avidity, a multivalent approach based on capturing the query protein, expressed as Fc-tagged ECD, on protein A-coated microbeads was developed4.
<ol>
	<li>Label the carrier IgG used for detection with Cy5 monoreactive dye and separate the free dye using desalting columns. Determine the dye to protein ratios by measuring ultraviolet absorbance at 280 and 650 nm. 
	Note: Protein-dye ratios between 2.0 and 4.0 are normally used. It is recommended to spin the Cy5 conjugates at 100,000 x g for 15 min to remove potential soluble aggregates due to the labeling process.</li>
	<li>Determine the optimal microbead-to-protein ratio by titration of protein A against a constant amount of the query protein. The minimal saturating volume of beads where no free Fc-tagged protein remains, as measured using a competitive assay determined by biolayer interferometry, is used for the screening.</li>
	<li>Form the microbead-protein complexes by incubating the Fc-tagged query and the Cy5-IgG with protein A microbeads and mix on a tube rotator in PBS for 30 min at room temperature protected from light.</li>
	<li>To form these complexes, use the query protein at a final concentration of 20 &#956;g/mL. 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 (150,000 Da) and multiply by 40 &#956;g/mL to determine the concentration of Cy5-IgG needed.</li>
	<li>Supplement samples with soluble protein A (1 mg/mL) immediately prior to incubation with the microarray slides (see 4.2 section below) to prevent binding of any free protein A beads to the Fc fusion proteins that may be present on the array. 
	Note: The final reaction volume may vary depending on the hybridization station or incubation chamber utilized. The instrumentation described in the Table of Materials allows sample incubation using relatively small volumes (~200 &#956;L per slide).</li>
</ol>
4. Extracellular Protein Microarray Screening and Processing.
Note: There are a number of manufacturers that provide automatic processing platforms. If a hybridization station is not available, the following steps can be performed manually, ensuring that there is sufficient volume of buffer to keep the slides submerged at all times.
<ol>
	<li>Warm the slides at room temperature and rinse with PBS/0.1% Tween 20 (PBST) to remove residual glycerol before loading onto the hybridization station.</li>
	<li>Use the following screening protocol:
	<ol>
		<li>Wash with PBST for 1 min.</li>
		<li>Load 200 &#956;L of 1 mg/mL protein A in PBS/5% milk and incubate for 30 min to prevent uncomplexed protein A microbeads from binding to Fc-tagged proteins that may be present in the microarray.</li>
		<li>Wash five times with PBST for 1 min.</li>
		<li>Load 200 &#956;L of the query:microbeads complex in the presence of 1 mg/mL protein A and incubate for 30 min.</li>
		<li>Wash with PBST for 1 min.</li>
	</ol>
	</li>
	<li>After hybridization, rinse slides with water, place in individual 50-mL conical tubes, and dry by spinning at 900 x g for 5 min.</li>
	<li>Finally, scan the slides with a microarray scanner appropriate to detect Cy3 (if BSA-Cy3 has been printed) and Cy5 fluorescence by exciting at 532 and 635 nm, respectively.</li>
	<li>Perform data analysis using the accompanying microarray data analysis. The relevant array list (as a .gal file) is loaded into the data extraction software. At this stage, utilize the Cy3-BSA spots to find blocks and use the auto-fitting options in the software, followed by manual alignment of the features when necessary. Save files as .gpr files for further analysis.</li>
</ol>
5. Data Analysis
<ol>
	<li>Save data as GPR files and process in R using the limma package, commonly utilized for analysis of microarray data4,5.
	<ol>
		<li>For preprocessing, do background correction and within-slide normalization. Since each protein is printed in duplicated spots, combine both replicate measurements to create a single score for that protein in the library.</li>
		<li>Calculate scores for each slide and analyze results for the intersection of high-scoring candidates between slides.</li>
		<li>Use duplicate microarrays from separate print-runs to control for slide variability and use intersection plots representing data from both screens to call final hits.</li>
		<li>Finally, perform an additional level of filtering to exclude promiscuous proteins within the array. Such non-specific binders are identified as proteins exceeding a specific threshold hit frequency as defined by the user across independent screens. 
		Note: This protocol is described in detail in Tom et al. 20135. In our case the non-specific binder calling is based on the determination of the cumulative prey hit rate, and a data-driven elimination threshold of 5% is used.</li>
	</ol>
	</li>
</ol>

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B.H., S.R-R and N.M-M. are Genentech employees and own shares in the Genentech Inc./Roche group.

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&amp;nbsp;</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>&amp;nbsp;009-000-003</td><td>Irrelevant IgG for labeling</td></tr><tr><td>Protein A</td><td>Sigma</td><td>&amp;nbsp;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 ...

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Research

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Biochemistry

7.1K Views.  Genentech. 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.

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

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

Genetics

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Biology

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Bioengineering

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

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 ...

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.

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 ...

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential

This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

Amplified Luminescent Proximity Homogeneous Assay: A Bead-Based Proximity Assay to Screen Small Molecules Inhibiting Protein-Protein Interactions

Source: Wang, L. et al., <a href="https://app.jove.com/v/62762">Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay.</a> J. Vis. Exp. (2021)
In this video, we demonstrate the amplified luminescent proximity homogeneous assay&#8212;a bead-based proximity assay. It utilizes homogeneously-sized acceptor and donor beads, immobilized with two proteins that tend to interact to screen potential small molecules inhibiting protein interactions.

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

Summary

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Chapters in this video

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

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

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Amplified Luminescent Proximity Homogeneous Assay: A Bead-Based Proximity Assay to Screen Small Molecules Inhibiting Protein-Protein Interactions