Name: An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation (Video) | JoVE Journal
Uploaded: 2022-06-06T13:00:00.000+00:00
Duration: 7 min 45 s
Description: Learn the optimized SiMPull assay for quantifying protein phosphorylation at the single-molecule level. This method improves antibody labeling and fixation while reducing autofluorescence for robust analysis.

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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

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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.

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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

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 ...

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 ...

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Research

JoVE Journal

Biochemistry

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

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This video demonstrates a sensitive quantification technique of protein phosphorylation using a single-molecule pull-down assay. The functionalization of polyethylene glycol-biotin and the use of labeled antibodies increases the detection of phosphorylated tyrosine with specificity.

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Oligopeptide Competition Assay for Phosphorylation Site Determination

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Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

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Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

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

Learn the optimized SiMPull assay for quantifying protein phosphorylation at the single-molecule level. This method improves antibody labeling and fixation while reducing autofluorescence for robust analysis.

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

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