Name: an optimized single-molecule pull-down assay for quantification of protein phosphorylation
Uploaded: 2022-06-06T13:00:00
Description: Watch this Scientific Journal Video about An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation at JoVE.com

This work was supported by the National Institutes of Health R35GM126934, R01AI153617, and R01CA248166 to DSL. EMB was supported through the ASERT-IRACDA program (NIH K12GM088021) and JAR by the UNM MARC program (NIH 2T34GM008751-20). We gratefully acknowledge using the University of New Mexico Comprehensive Cancer Center fluorescence microscopy shared resource, supported by NIH P30CA118100. We want to acknowledge Drs. Ankur Jain and Taekijip Ha, whose original development of SiMPull inspired this work. 
ES-C present address: Immunodynamics Group, Laboratory of Integrative Cancer Immunology, Center for Cancer Research, National Cancer Institute, Bethesda

1. Coverslip preparation
NOTE: For this step, one needs to wear personal protective equipment (PPE), which includes a double layer of nitrile gloves, safety glasses or face shield, and a lab coat.
<ol>
	<li>Perform piranha etching to remove organic debris from the glass. 
	CAUTION: Piranha solution is a strong oxidizing agent that is corrosive and highly reactive when in contact with organic materials. Reaction with organic debris is exothermic and potentially explosive...

<ol>
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The protocol described here was optimized to enable quantitative measurements of receptor phosphorylation at the single protein level. Several straightforward but important modifications to the SiMPull protocol were developed that improved the reliability of the measurement for phospho-tyrosine detection, including reduction of autofluorescence with NaBH4 treatment and postfixing of the sample to prevent antibody dissociation. Using the green channel mask to identify receptor locations for calculation of coloc...

Membrane-associated signaling is tuned by a combination of ligand-induced membrane receptor activation and recruitment of downstream accessory proteins that propagate the signal. Phosphorylation of key tyrosines in receptor cytoplasmic tails is critical to initiating the formation of signaling complexes, or signalosomes1,2. Therefore, an important question in biology is how phosphorylation patterns are created and maintained to recruit signaling partners and dictate cellular outcomes. This includes understanding the heterogeneity of receptor phosphorylation, both in abundance and in the specific phosphotyrosine patterns that can provide a means of manipulating signaling outputs by dictating the composition of the signalosome3,4,5,6,7. However, there are limitations in current methods to interrogate protein phosphorylation. Western blot analysis is excellent for describing trends of protein phosphorylation but is semi-quantitative8 and does not provide information on the heterogeneity of the system because thousands to millions of receptors are averaged together. While western blots allow probing a sample using phospho-specific antibodies to specific tyrosines, they cannot provide information on multisite phosphorylation patterns within the same protein. Quantitative phosphoproteomics report on phosphotyrosine abundance, but there are limitations to detecting multisite phosphorylation, as the residues of interest need to be located within the same peptide (typically 7-35 amino acids) that is generated by enzymatic digestion9,10,11.
To overcome the limitations mentioned above, the single-molecule pull-down (SiMPull) assay has been adapted to quantify the phosphorylation states of intact receptors at the single-molecule level. SiMPull was first demonstrated as a powerful tool for interrogating macromolecular complexes by Jain et al.12,13. In SiMPull, macromolecular complexes were immunoprecipitated (IP) on antibody-functionalized glass coverslips and then analyzed through single-molecule microscopy for protein subunit number and co-IP with complex components12. A modification by Kim et al.14, termed SiMBlot, was the first to use a variation of SiMPull to analyze phosphorylation of denatured proteins.&#160;The SiMBlot protocol relies on capturing biotinylated cell surface proteins using NeutrAvidin-coated coverslips, which are then probed for phosphorylation with phospho-specific antibody labeling14. Despite these advances, improvements were needed to make the quantification of posttranslational modification more robust and applicable to a broader range of proteins.
The present protocol describes an optimized SiMPull approach that was used to quantify phosphorylation patterns of intact epidermal growth factor receptor (EGFR) in response to a range of ligand conditions and oncogenic mutations15. While this work focuses on EGFR, this approach can be applied to any membrane receptor and cytosolic proteins of interest (POI), for which quality antibodies are available. The protocol includes steps to reduce sample autofluorescence, a sample array design that requires minimal sample volume with simultaneous preparation of up to 20 samples, and optimization of antibody labeling and fixation conditions. Data analysis algorithms have been developed for single-molecule detection and quantification of phosphorylated proteins.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>MTC Bio</td>
			<td>C2000</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Tris-HCl pH 7.4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Tris-HCl pH 8.0/ 50 mM NaCl</td>
			<td></td>
			<td></td>
			<td>T50 Buffer</td>
		</tr>
		<tr>
			<td>100 mm Tissue Culture dish</td>
			<td>CELLTREAT</td>
			<td>229620</td>
			<td>Storage of piranha etched glass/arrays</td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td></td>
			<td></td>
			<td>Functionalized Glass storage/ KOH reuse</td>
		</tr>
		<tr>
			<td>50 mM Tris-HCl pH 7.2/150 mM NaCl</td>
			<td></td>
			<td></td>
			<td>Lysis Buffer Component</td>
		</tr>
		<tr>
			<td>60 mm Tissue Culture dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>8% Glutaraldehyde Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>16020</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Acetone (C3H6O)</td>
			<td>Millipore Sigma</td>
			<td>270725</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 NHS Ester</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-20006</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human EGFR (External Domain) &#8211; Biotin</td>
			<td>Leinco Technologies, Inc</td>
			<td>E101</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p-Tyr Antibody (PY99) Alexa Fluor 647</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-7020 AF647</td>
			<td></td>
		</tr>
		<tr>
			<td>Bath-sonicator</td>
			<td>Branson</td>
			<td>1200</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Pierce</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-PEG</td>
			<td>Laysan Bio</td>
			<td>Biotin-PEG-SVA, MW 5,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Gold Biotechnology</td>
			<td>A-420-1</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Buchner funnel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Millipore Sigma</td>
			<td>C4901</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>Bioworld</td>
			<td>30900017-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Filtering Flask</td>
			<td>Fisher Scientific</td>
			<td>S15464</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin Jar</td>
			<td>WHEATON</td>
			<td>900470</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips 24 x 60 #1.5</td>
			<td>Electron Microscopy Sciences</td>
			<td>63793</td>
			<td></td>
		</tr>
		<tr>
			<td>DipImage</td>
			<td></td>
			<td></td>
			<td>https://diplib.org/</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Caisson Labs</td>
			<td>DML19-500</td>
			<td></td>
		</tr>
		<tr>
			<td>emCCD camera</td>
			<td>Andor iXon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Optima</td>
			<td>Bio-Techne</td>
			<td>S12450H</td>
			<td>Heat Inactivated</td>
		</tr>
		<tr>
			<td>Fusion 360 software</td>
			<td>Autodesk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin G418 Disulfate</td>
			<td>Caisson Labs</td>
			<td>G030-5GM</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid (CH3COOH)</td>
			<td>JT Baker</td>
			<td>JTB-9526-01</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Glass serological pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Stir Rod</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (D-(+)-Glucose)</td>
			<td>Millipore Sigma</td>
			<td>D9434</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Halt Phosphotase and Protease Inhibitor Cocktail (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>78446</td>
			<td>Lysis Buffer Component</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore Sigma</td>
			<td>H3375</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid (HCl)</td>
			<td>VWR</td>
			<td>BDH7204-1</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (H2O2) (3%)</td>
			<td></td>
			<td>HX0645</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (H2O2) (30%)</td>
			<td>EMD Millipore</td>
			<td>HX0635-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630 (NP-40)</td>
			<td>Sigma Aldrich</td>
			<td>I8896</td>
			<td>Lysis Buffer Component</td>
		</tr>
		<tr>
			<td>ImmEdge Hydrophobic Barrier Pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersol 518F immersion oil</td>
			<td>Zeiss</td>
			<td>444960-0000-000</td>
			<td></td>
		</tr>
		<tr>
			<td>in-house vacuum line</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-164</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnessium Chloride Hexahydrate (MgCl2-6H2O)</td>
			<td>MPBio</td>
			<td>2191421</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td></td>
			<td>Curve Fitting Toolbox, Parallel Computing Toolbox, and Statistics and Machine Learning toolbox</td>
		</tr>
		<tr>
			<td>Methanol (CH3OH)</td>
			<td>IBIS Scientific</td>
			<td>MX0486-1</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Milli-Q water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mix-n-Stain CF Dye Antibody Labeling Kits</td>
			<td>Biotium</td>
			<td>92245</td>
			<td>Suggested conjugation kit</td>
		</tr>
		<tr>
			<td>mPEG</td>
			<td>Laysan Bio</td>
			<td>mPEG-succinimidyl valerate, MW 5,000</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(2-aminoethyl)-3-aminopropyltrimethoxysilane</td>
			<td>UCT United Chemical</td>
			<td>A0700</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Nanogrid</td>
			<td>Miraloma Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NeutrAvidin Biotin Binding Protein</td>
			<td>Thermo Fisher Scientific</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen (compressed gas)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NVIDIA GPU with CUDA</td>
			<td></td>
			<td></td>
			<td>Look for compatibility at https://www.mathworks.com/help/parallel-computing/gpu-support-by-release.html</td>
		</tr>
		<tr>
			<td>Olympus iX71 Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M Sealing Film</td>
			<td>The Lab Depot</td>
			<td>HS234526C</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7.4</td>
			<td>Caisson Labs</td>
			<td>PBL06</td>
			<td></td>
		</tr>
		<tr>
			<td>PC-200 Analog Hot Plate</td>
			<td>Corning</td>
			<td>6795-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-EGF Receptor (Tyr1068) (1H12) Mouse mAb</td>
			<td>Cell Signaling Technology</td>
			<td>2236BF</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Millipore Sigma</td>
			<td>529552</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide (KOH)</td>
			<td>Millipore Sigma</td>
			<td>1050330500</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Premium PLA Filament, 1.75 mm diameter</td>
			<td>Raise 3D</td>
			<td>PMS:2035U/RAL:3028</td>
			<td>Printing temperature range: 205-235 &#176;C</td>
		</tr>
		<tr>
			<td>Pro2 3D printer</td>
			<td>Raise 3D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex 1 L beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PYREX 100 mL storage bottles</td>
			<td>Corning</td>
			<td>1395-100</td>
			<td>CH3OH/C3H6O reuse</td>
		</tr>
		<tr>
			<td>Pyrex 250 mL beakers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex 4 L beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quad-view Image Splitter</td>
			<td>Photometrics</td>
			<td>Model QV2</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>EP-5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>RevCount Cell Counters, EVE Cell Counting Slides</td>
			<td>VWR</td>
			<td>10027-446</td>
			<td></td>
		</tr>
		<tr>
			<td>Semrock emission filters: blue (445/45 nm), green (525/45 nm), red (600/37 nm), far-red (685/40 nm)</td>
			<td>Semrock</td>
			<td>LF405/488/561/635-4X4M-B-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>smite single molecule analysis package</td>
			<td></td>
			<td></td>
			<td>https://github.com/LidkeLab/smite.git</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Sigma Aldrich</td>
			<td>S6014</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Sodium Borohydride (NaBH4)</td>
			<td>Millipore Sigma</td>
			<td>452874</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Millipore Sigma</td>
			<td>S9625</td>
			<td>Activate by successive heat and pH cycling</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>VWR</td>
			<td>BDH3247-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Orthovanadate (Na3VO4)</td>
			<td>Millipore Sigma</td>
			<td>S6508</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Sulfuric Acid (H2SO4)</td>
			<td>Millipore Sigma</td>
			<td>258105</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>TetraSpeck Microspheres</td>
			<td>Thermo Fisher Scientific</td>
			<td>T7279</td>
			<td>multi-fluorescent beads</td>
		</tr>
		<tr>
			<td>Tris (Trizma) base</td>
			<td>Millipore Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain, 0.4%</td>
			<td>Thermo Fisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.05%</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300120</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized single-molecule pull-down assay for quantification of protein phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

A cartoon depicting the SiMPull process is shown in Figure 1A. Coverslips are functionalized using NeutrAvidin as an anchor for biotinylated anti-EGFR antibodies to capture EGFR-GFP from total protein lysates. After washing away unbound protein, the phosphorylated receptors are labeled with an anti-phosphotyrosine (anti-PY) antibody15. Figure 1B shows an image of the hydrophobic array, where multiple samples can be prepared and imaged on ...

Watch this Scientific Journal Video about An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation at JoVE.com

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

Research

JoVE Journal

Biochemistry

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

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Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

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Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

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Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

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Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...