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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

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

Published: January 7th, 2019

DOI:

10.3791/58451

1Receptor Discovery group, Microchemistry, Proteomics and Lipidomics Department, Genentech, 2Portfolio Management and Operations, 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.

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.

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, sign....

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1. Generation of a Library of Extracellular Human Proteins

  1. 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.
  2. For cell surface receptors, determine the extracellular domain (ECD) boundaries by identifying the signal peptide and transmembrane regions using software tools. Some o.......

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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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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.......

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

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

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