Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Summary

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Abstract

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Introduction

Dynamic protein-protein interactions constitute the molecular signaling cascades and motile structures that are the functional basis of most cellular physiology¹. These processes are often depicted as linear signaling pathways that switch between steady states based on single inputs, but experimental and modeling data clearly show that they function as integrated networks^2,3,4. In the case of G proteins, different receptors often have the ability to activate the same G protein, and a single receptor can also activate more than one type of G protein

Protocol

1. Assay design

1. Candidate antibody preparation
   1. For each protein of interest, choose 3 to 5 antibodies to screen. When possible, use monoclonal antibodies that recognize different epitopes. Also include one non-specific control antibody.
   2. To remove Tris, perform buffer exchange by adding the antibody to a 30 kDa spin filter, spinning down to its minimum volume, adding 500 µL of phosphate-buffered saline (PBS), and repeating 3 times. To remove carrier proteins, perform antibody purification according to the manufacturer's protocol (see Table of Materials for specific purification recommendation).
      NOTE: ....

Results

Antibody Screening
Figure 2B shows the results of a screen for the protein Connexin36. Most IP_probe combinations produce no signal over IgG controls. IP with the monoclonal antibody 1E5 and probe with either 1E5 or the polyclonal antibody 6200 produces a rightward shift in the bead distribution compared to IgG controls. Here, IP 1E5 and probe 6200poly were selected to avoid using the same antibody as IP and probe, both to reduce the probability of a non.......

Discussion

The QMI assay requires substantial investment in antibody panel development, equipment and reagents, but once the assay is established, one can collect high-dimensional data observing protein interaction networks as they respond to experimentally-controlled stimuli. Technically, QMI requires careful pipetting and tracking of sample and antibody well locations. Carefully labeling the assay plates is useful, as is making a detailed template of well locations on paper, which is then saved for data analysis. The importance o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well flat bottomed plates	Bio Rad	171025001
96-well PCR plates	VWR	82006-704
Bioplex 200 System with HTF	Bio Rad	171000205	modiefied to keep partially refrigerated, see Figure S1 for details
Bio-Plex Pro Wash Station	Bio Rad	30034376
BSA	Sigma
CML beads	Invitrogen	C37481
EDTA	Sigma	E6758
EZ-Link Sulfo-NHS-Biotin	Thermo Scientific	A39256
MagPlex Microspheres	Luminex	MC12xxx-01	xxx is the 3 digit bead region
Melon Gel IgG Spin Purification Kit	Thermo Scientific	45206	used for antibody purification
MES	Sigma	M3671
Microplate film, non-sterile	USA Scientific	2920-0000
Phosphotase inhibitor cocktail #2	Sigma	P5726
Protease inhibitor cocktail	Sigma	P8340
Sandwich Prep Refrigerator	Norlake	SMP 36 15	for custom refrigeration of Bioplex 200
Sodium fluoride	Sigma	201154
Sodium orthovanadate	Sigma	450243
Streptavidin-PE	BioLegend	405204
Sulfo NHS	Thermo Scientific	A39269
Tris	Fisher Scientific	BP152