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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Discussion

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.......

Materials

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