Name: an optimized single-molecule pull-down assay for quantification of protein phosphorylation
Uploaded: 2022-06-06T13:00:00.000+00:00
Duration: 7 min 45 s
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

<ol>
	<li>Lemmon, M. A., Schlessinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Signaling+by+Receptor+Tyrosine+Kinases.">Cell Signaling by Receptor Tyrosine Kinases.</a> Cell. 141 (7), 1117-1134 (2010).</li><li>Seet, B. T., Dikic, I., Zhou, M. M., Pawson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reading+protein+modifications+with+interaction+domains.">Reading protein modifications with interaction domains.</a> Nature Reviews Molecular Cell Biology. 7 (7), 473-483 (2006).</li><li>Coba, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotransmitters+drive+combinatorial+multistate+postsynaptic+density+networks.">Neurotransmitters drive combinatorial multistate postsynaptic density networks.</a> Science Signaling. 2 (68), (2009).</li><li>Gibson, S. K., Parkes, J. H., Liebman, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+modulates+the+affinity+of+light-activated+rhodopsin+for+g+protein+and+arrestin.">Phosphorylation modulates the affinity of light-activated rhodopsin for g protein and arrestin.</a> Biochemistry. 39 (19), 5738-5749 (2000).</li><li>Stites, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+mechanistic+models+to+integrate+and+analyze+multiple+proteomic+datasets.">Use of mechanistic models to integrate and analyze multiple proteomic datasets.</a> Biophysical Journal. 108 (7), 1819-1829 (2015).</li><li>Hause, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+binary+interaction+mapping+of+SH2+domains+via+fluorescence+polarization+reveals+novel+functional+diversification+of+ErbB+receptors.">Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional diversification of ErbB receptors.</a> PLOS ONE. 7 (9), 44471 (2012).</li><li>Lau, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+encoding+of+the+effect+of+a+partial+agonist+on+individual+opioid+receptors+by+multisite+phosphorylation+and+threshold+detection.">Quantitative encoding of the effect of a partial agonist on individual opioid receptors by multisite phosphorylation and threshold detection.</a> Science Signaling. 4 (185), (2011).</li><li>Mishra, M., Tiwari, S., Gomes, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+purification+and+analysis:+next+generation+Western+blotting+techniques.">Protein purification and analysis: next generation Western blotting techniques.</a> Expert Review of Proteomics. 14 (11), 1037-1053 (2017).</li><li>Brunner, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+multiple+fragmentation+methods+on+an+orbitrap+fusion+for+top-down+phospho-proteoform+characterization.">Benchmarking multiple fragmentation methods on an orbitrap fusion for top-down phospho-proteoform characterization.</a> Analytical Chemistry. 87 (8), 4152-4158 (2015).</li><li>Swaney, D. L., Wenger, C. D., Coon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Value+of+using+multiple+proteases+for+large-scale+mass+spectrometry-based+proteomics.">Value of using multiple proteases for large-scale mass spectrometry-based proteomics.</a> Journal of Proteome Research. 9 (3), 1323-1329 (2010).</li><li>Curran, T. G., Zhang, Y., Ma, D. J., Sarkaria, J. N., White, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MARQUIS:+A+multiplex+method+for+absolute+quantification+of+peptides+and+posttranslational+modifications.">MARQUIS: A multiplex method for absolute quantification of peptides and posttranslational modifications.</a> Nature Communications. 6 (1), 1-11 (2015).</li><li>Jain, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+cellular+protein+complexes+using+single-molecule+pull-down.">Probing cellular protein complexes using single-molecule pull-down.</a> Nature. 473 (7348), 484-488 (2011).</li><li>Jain, A., Liu, R., Xiang, Y. K., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+pull-down+for+studying+protein+interactions.">Single-molecule pull-down for studying protein interactions.</a> Nature Protocols. 7 (3), 445-452 (2012).</li><li>Kim, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pairwise+detection+of+site-specific+receptor+phosphorylations+using+single-molecule+blotting.">Pairwise detection of site-specific receptor phosphorylations using single-molecule blotting.</a> Nature Communications. 7 (1), 1-10 (2016).</li><li>Salazar-Cavazos, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisite+EGFR+phosphorylation+is+regulated+by+adaptor+protein+abundances+and+dimer+lifetimes.">Multisite EGFR phosphorylation is regulated by adaptor protein abundances and dimer lifetimes.</a> Molecular Biology of the Cell. 31 (7), 695 (2020).</li><li>Huyer, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+inhibition+of+protein-tyrosine+phosphatases+by+vanadate+and+pervanadate.">Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate.</a> Journal of Biological Chemistry. 272 (2), 843-851 (1997).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150 (1), 76-85 (1985).</li><li>Keller, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Objective+lenses+for+confocal+microscopy.">Objective lenses for confocal microscopy.</a> Handbook of Biological Confocal Microscopy. , 145-161 (2006).</li><li>Hendriks, C. L. L., van Vliet, L. J., Rieger, B., van Kempen, G. M. P., van Ginkel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dipimage: a scientific image processing toolbox for MATLAB. , (1999).</li><li>fitgeotrans: Fit geometric transformation to control point pairs. The MathWorks Inc Available from: <a target="_blank" href="https://www.mathworks.com/images/ref/fitgeotrans.html">https://www.mathworks.com/images/ref/fitgeotrans.html</a> (2013)</li><li>Raghavachari, N., Bao, Y. P., Li, G., Xie, X., M&#252;ller, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+autofluorescence+on+DNA+microarrays+and+slide+surfaces+by+treatment+with+sodium+borohydride.">Reduction of autofluorescence on DNA microarrays and slide surfaces by treatment with sodium borohydride.</a> Analytical Biochemistry. 312 (2), 101-105 (2003).</li><li>Wang, X., Park, S., Zeng, L., Jain, A., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+single-cell+single-molecule+pull-down.">Toward single-cell single-molecule pull-down.</a> Biophysical Journal. 115 (2), 283-288 (2018).</li><li>Chandradoss, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+passivation+for+single-molecule+protein+studies.">Surface passivation for single-molecule protein studies.</a> Journal of Visualized Experiments. (86), e50549 (2014).</li></ol>

The authors have nothing to disclose.

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><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 (C&lt;sub&gt;3&lt;/sub&gt;H&lt;sub&gt;6&lt;/sub&gt;O)</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) &amp;ndash; 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&#039;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 (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Millipore Sigma</td><td>C4901</td><td>Tyrode&#039;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 (CH&lt;sub&gt;3&lt;/sub&gt;COOH)</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&#039;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&#039;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 (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;) (3%)</td><td></td><td>HX0645</td><td></td></tr><tr><td>Hydrogen Peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;) (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 (MgCl&lt;sub&gt;2&lt;/sub&gt;-6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>MPBio</td><td>2191421</td><td>Tyrode&#039;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 (CH&lt;sub&gt;3&lt;/sub&gt;OH)</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&#039;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 &amp;deg;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>CH&lt;sub&gt;3&lt;/sub&gt;OH/C&lt;sub&gt;3&lt;/sub&gt;H&lt;sub&gt;6&lt;/sub&gt;O 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 (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>S6014</td><td>Hazardous</td></tr><tr><td>Sodium Borohydride (NaBH&lt;sub&gt;4&lt;/sub&gt;)</td><td>Millipore Sigma</td><td>452874</td><td>Tyrode&#039;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 (Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Millipore Sigma</td><td>S6508</td><td>Hazardous</td></tr><tr><td>Sulfuric Acid (H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</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 ...

an-optimized-single-molecule-pull-down-assay-for-quantification

Research

JoVE Journal

Biochemistry

2.8K Views.  University of New Mexico School of Medicine. The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

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

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Biology

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Immunology and Infection

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

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

Summary

Explore More Videos

Chapters in this video

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

Assaying Protein Kinase Activity with Radiolabeled ATP

Oligopeptide Competition Assay for Phosphorylation Site Determination

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Identification of Post-translational Modifications of Plant Protein Complexes