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 colocalization with the anti-PY antibody also improves the measurement accuracy by removing potential artifacts from the non-specific binding of the antibody to the cell lysate. The two-color imaging was utilized to detect the fraction of receptors phosphorylated. In this scenario, the receptor was genetically tagged with GFP, and the antibody was directly labeled with a far-red dye. The SiMPull approach applies to other protein targets for which specific antibodies are available, including intracellular proteins. In addition, because denaturing conditions are not required, multisubunit receptors/complexes can also be captured. However, denaturation may be incorporated if the PTMs of interest are located in structured regions of the protein14. Ultimately, SiMPull can be readily expanded to include simultaneous labeling of distinct phospho-tyrosines on individual receptors to quantify multisite phosphorylation patterns15. The interrogation of full-length, intact receptors in such a way cannot be achieved by other standard methods, including western blotting and phospho-mass spectrometry.
Along with the advantages of SiMPull, some limitations need to be considered. As with any antibody-based technique, the affinity and specificity of antibodies used are critical to the success of the measurement. Therefore, it is important to optimize antibody labeling conditions and ideally avoid secondary antibodies by using directly-labeled primary antibodies. Furthermore, the surface-bound antibodies will precipitate proteins localized to the plasma membrane and within cytosolic compartments. This can underestimate phosphorylation since cytosol-localized proteins are not accessible to the exogenously added ligand. Extra steps must be taken to correct the receptor surface levels (step 6.2.10). The anti-phosphotyrosine antibodies exhibited some non-specific binding once lysate was present. To avoid this artifact, the EGFR was genetically-tagged with GFP to identify the location of receptors, which allowed us to exclude the anti-PY signal off the receptor. If endogenous proteins are to be interrogated, then counterstaining with a total protein antibody can provide the mask image, with appropriate correction for any non-specific binding. Finally, while SiMPull provides information on heterogeneity at the protein level, the lysate generated in this protocol is from thousands of cells, and cell-to-cell variability is lost. However, advances towards single-cell SiMPull have been made using a flow chamber consisting of a coverslip and a microscope side with a 10 &#181;m gap; bacteria were sparsely plated on the coverslip while the slide was functionalized with antibody to capture the desired proteins. Upon lysis of the bacteria, the proteins from each cell were captured in a confined area on the antibody-coated slide22. Similar single-cell SiMPull analysis of mammalian cells and protein phosphorylation may be possible in the future.
The SiMPull protocol contains several critical steps required to ensure high-quality data. For example, the protocol includes an elaborate preparation of the coverslip glass. Piranha etching coverslips thoroughly cleans the glass and increases hydroxyl groups and hydrophilicity, which are needed to optimize the coverslip surface. Following several washes with organic solvents, KOH treatment provides additional hydroxyl groups for aminosilanization13,23, which coats the glass with amine groups for PEG and biotin-PEG binding. Improper cleaning or functionalization at any of these steps will interfere with protein pull-down. Control of the molar ratio of PEG:biotin-PEG, along with lysate concentration, are key factors in obtaining appropriate protein IP density on the SiMPull substrate. As with any biological assay, there is variability between cell lysate preparations, and small differences between phosphorylation percentages may be seen between sample replicates. Therefore, it is important to measure the phosphorylation levels of different tyrosine sites within the same sample. The sample chamber described in this protocol provides a system to collect many data points in one imaging session and allows for averaging over multiple SiMPull experiments.
On the image acquisition side, it is important to obtain the fiducial sample to ensure an accurate channel overlay; otherwise, colocalization will not be accurate. It is also important to optimize laser power and camera settings to maximize signal-to-noise while at the same time minimizing photobleaching. Lastly, while the sample array requires a small amount of sample and reagents, the low volumes are susceptible to evaporation during the imaging session. It is important to periodically check the sample array (~30-45 min) and add buffer as needed to prevent samples from drying.
The present protocol demonstrated the use of SiMPull to quantify membrane receptor phosphorylation states. While focused on EGFR, the approach can be applied to other cell surface receptors and intracellular proteins and protein complexes, as long as appropriate antibodies are available. Another potential use for SiMPull is to interrogate the contents and phosphorylation status of phase-separated condensates. In addition, SiMPull can be used to measure other PTMs, such as ubiquitination. Therefore, SiMPull provides a unique tool for cell biologists to interrogate PTMs on intact proteins and correlate PTM patterns with cellular outcome.

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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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 the same coverslip. One advantage of this sample holder is that minimal sample volumes of ~10 &#181;L are required. The coverslip can be imaged by placing it directly on the microscope stage. However, it is helpful to stabilize the coverslip by using a coverslip holder. The coverslip holder shown in Figure 1B was created using a 3D printer, and the blueprint is provided in Supplementary Coding File 5. The autofluorescence of the hydrophobic ink is a useful guide to finding the focal plane of the sample (Figure 1C). An example of a multichannel raw image is shown in Figure 1D. An overlay of the raw green and far-red channels is shown in Figure 1E.
Figure 2&#160;outlines the analysis workflow and provides representative data. Data acquisition first starts with acquiring fiducials for channel registration, which is used to overlay the individual spectral channels data (Figure 2A). Bright-field images are taken using a nanogrid pattern that passes white light and is detected in each spectral channel of the image splitter (not shown). The green channel acts as the reference channel, and the far-red channel is the shifted channel. The local weighted mean transform is calculated using the fitgeotrans20 function in MATLAB and is used to shift far-red coordinates into the coordinate frame of the green channel. This transform uses a second-order polynomial model at every control point. Multichannel data of the SiMPull array is then acquired. This workflow consisted of a semi-automated acquisition, where a starting ROI was selected for the specific sample square, and three regions around this area were imaged, such that each dataset contains the full quad-view image from three independent ROIs (Figure 1D). In each spectral channel, the emitter candidate locations are found by applying a difference of Gaussian filter to images and identifying local maxima. Subregions (boxes, Figure 2B) are drawn around local maxima, and emitter photon counts are estimated by assuming each subregion contains only one emitter. Subregions holding emitter candidates with photon counts above a minimum value are retained for fitting. A Gaussian point spread function (PSF) fits each emitter candidate within small subregions roughly centered around each emitter. The resulting localizations are thresholded based on their photon count, background, Cram&#233;r-Rao lower bound of the fit coordinates, PSF variance (i.e., PSF width), and a p-value describing the goodness of fit of the PSF model. A Gaussian image is created for each spectral channel, with uniform intensity Gaussian blobs placed at the coordinates for each good fit (Figure 2C). Colocalization is visualized by overlaying the Gaussian images from each spectral channel using the transform calculated from the fiducial sample (Figure 2D). It is important to fluorescently label the receptor for identification since there is still non-specific binding of the anti-phosphotyrosine antibodies to the surface when cell lysate is present. The EGFR-GFP (green channel) is used to generate a mask of the receptor locations, and only the AF647-anti-PY signal (far-red channel) within that mask is counted (Figure 2D). Pairs within 1 pixel (106.7 nm pixel size) are considered to be colocalized and saved to a list containing the reference channel coordinates. The percentage of AF647 colocalized with GFP is calculated to determine the fraction of phosphorylated receptors (Figure 2E).
There are several critical steps to ensure good data quality. One such effort is to incubate the coverslip array with NaBH4 as described in the protocol to quench autofluorescence in the green channel. This autofluorescence refers to the non-specific signal due to possible impurities on the glass, containing single or conjugated &#960; bonds21. Such impurities are potentially from the aminosilane and PEG reagents used in the functionalization process or dust from the air, and tend to fluoresce in the green spectral channel. Despite efforts to keep glass stored under nitrogen, these molecules may also be generated through oxidation that occurs in storage. NaBH4 has also been used to reduce fluorescence from impurities on slides and microarrays, including those with silane coating16. Figure 3A shows the reduction in the number of background detections that occur when the piranha etched glass is treated with NaBH4. While NaBH4 reduces background fluorescence dramatically, some emitters are still detected in the green channel. One can correct this by acquiring background images from lysate-free samples (Figure 3D) and subtracting the average number of background localizations from the GFP-containing samples. Fluorescence from impurities was not detected in the far-red channel. If the receptor density is too high, multiple GFP emitters can be found within a single diffraction-limited spot (data not shown). Using step-photobleaching to identify the number of GFPs per spot, we found that a receptor density between 0.04-0.08 proteins/&#181;m2 provided sufficient spacing between single emitters to remove the potential of finding multiple emitters per spot12. The receptor density can be optimized by varying the amount of IP antibody bound to the glass surface or the amount of lysate added. It is critical to ensure that the antibody targeting the POI is used at saturating levels. It is recommended to acquire an antibody concentration curve on phosphorylated samples to determine the appropriate labeling conditions (Figure 3B). In addition, the phospho-specificity of an antibody needs to be validated with resting samples and/or treatment with protein-specific kinase inhibitors (Figure 3B). Antibodies will dissociate from the receptor during the imaging time window. Treating the sample with a combination of PFA and GA prevented signal loss (Figure 3C).
Finally, it is important to optimize the single-molecule fitting parameters. The first &#34;box finding&#34; step that identifies potential emitter candidates (Figure 2B) needs to be generous to allow many candidates to undergo the Gaussian Fitting. Thus, the minimum photon threshold for box finding can be relatively low to capture all real emitters and some background spots. It is also important to not set the box size and overlap allowance too small. Keeping the box size within 5-7 pixels and allowing two-pixel overlap is ideal for emitters at the recommended density. After box finding, the minimum photons threshold in the fitting step needs to be optimized. The minimum photons parameter contributes to determining which Gaussian fitted emitters pass as a true fit. To determine the proper minimum photon threshold for true GFP fits, the code includes a histogram plotting function to examine the photons/localization in both background (no cell lysate) and GFP-containing (plus cell lysate) samples (Figure 3D). This step is important because, while NaBH4 reduces the amount of fluorescence from impurities, it does not remove all background localizations. Figure 3D demonstrates the need to set a minimum photon threshold to reduce the number of detections from impurities. To determine this threshold, a histogram of background emitter intensities is calculated from imaging a sample that is not exposed to cell lysate (Figure 3D, top left). The majority of the background emitters were found to have values less than 475 photons. In comparison, the sample containing true GFP emitters showed a significant fraction of the distribution above 475 (Figure 3D, top right). The threshold is chosen by inspection to remove as many background counts as possible while minimizing the amount of signal loss from the lysate sample (Figure 3D, bottom row). The remaining background count density at this threshold is accounted for in the quantitative analysis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63665/63665fig01.jpg" /> 
Figure 1: Overview of sample preparation. (A) Cartoon depicting the SiMPull approach. Coverslips are functionalized with an antibody that recognizes the POI to capture that POI from whole cell lysates. The glass is first coated with PEG and biotin-PEG. NeutrAvidin is then bound to the biotin-PEG and acts as an anchor for the biotinylated anti-POI antibody. Phosphorylated proteins are then detected with a fluorescently labeled anti-PY antibody. (B) Photograph of the coverslip holder (red) with coverslip array in place and mounted on the microscope stage. The multisample arrays are generated using hydrophobic ink to create up to 20 individual sample squares on a single glass coverslip. The coverslip is 60 mm x 24 mm. (C) Example images of the hydrophobic ink autofluorescence (magenta) and fluorescent beads (green). The autofluorescence of the hydrophobic ink is a useful guide to find the focal plane at the coverslip surface. (D) Example of a raw data image with spectral channels separated on the camera chip by the Quad-view image splitter. The Quad-view filter set includes the following emission filters: blue (445/45 nm), green (525/45 nm), red (600/37 nm), far-red (685/40 nm). (E) Raw overlay of green and far-red channels. The white box indicates the region further examined in Figure 2B-D. Scale bar (C-E) = 2 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63665/63665fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63665/63665fig02.jpg" /> 
Figure 2: Data analysis workflow. (A) Channel registration is first performed on images acquired from the nanogrid. After cropping the two spectral channels of interest (here, green and far-red), the fiducial images for each channel are overlaid (left). Enlargement of the box in the left image (Inset) shows that the images are not yet truly registered. The emitters in each channel fit a Gaussian model and are localized (Registration). Localization of emitters is shown as circles for the far-red channel and crosses for the green channel. The final step is to apply a local weighted mean transform to shift the far-red channel localization coordinates into the green channel reference frame (Aligned). The calculated local weighted mean transform is then used to register the subsequent SiMPull data. (B) Representative images of the green/EGFR-GFP channel and the far-red/AF647-anti-PY channel. Single emitters above the background photon count are identified and marked with boxes. (C) The emission profile within each selected box is fit to a Gaussian model, and the emitters that fit the model of a single fluorophore PSF are kept. (D) A mask is created from the GFP emitters to identify the location of EGFR-GFP (green). Colocalization of EGFR-GFP and AF647-anti-PY identifies phosphorylated receptors (white). (E) The fraction of phosphorylated receptors is calculated from the colocalized EGFR-GFP and AF647-anti-PY fits. The bar graph compares PV + EGF treatment to resting cells, averaged for multiple measurements. Error bars represent standard error calculated assuming a binomial distribution. Scale bar = 2 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63665/63665fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63665/63665fig03.jpg" /> 
Figure 3: Critical steps to ensure data quality. (A) From left to right, the first three panels are representative images of the autofluorescence on glass under the respective conditions: after piranha etching, with PEG, and PEG plus NaBH4 treatment (indicated with +). Additionally, surface functionalization is retained after NaBH4 treatment as demonstrated by minimal non-specific PY99-AF647 binding while retaining robust binding of EGFR-GFP from the lysate. (B) A saturation curve must be acquired for each batch of antibodies used to ensure optimal antibody labeling. This figure shows the concentration curve for labeling EGFR with the site-specific phosphotyrosine antibody, anti-EGFR-pY1173. Minimal phosphorylation is detected in untreated cells (Resting, gray diamond). As a control for non-specific binding, cells were also treated with the EGFR kinase inhibitor, Lapatinib, before adding 100 nM of EGF (magenta triangle), which shows the expected prevention of EGFR phosphorylation. Error bars represent standard error assuming a binomial distribution. (C) Fixation of the sample with a combination of PFA and GA prevents antibody dissociation over time. Error bars represent standard error assuming a binomial distribution. (D) False positives are excluded by selecting the appropriate threshold for Gaussian fitting. Comparing the histogram of fit intensities at a low threshold (Threshold = 0; top) between background (no lysate) and real data (plus cell lysate) allows for selection of appropriate value (Threshold = 475; bottom) to remove fits from autofluorescent spots in the green channel. The vertical magenta line indicates a 475 photon threshold. Histograms are calculated from the same number of ROIs for each sample type (n = 3). Scale bar = 2 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63665/63665fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Coding File 1: zip file containing scripts and utilities for running SiMPull analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63665/Supplemental File 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 2: zip file containing the smite single-molecule analysis package. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63665/Supplemental File 2.zip" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 3: zip file containing the sample data. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63665/Supplemental File 3.zip" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 4: zip file containing representative sample data analysis outputs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63665/Supplemental File 4.zip" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 5: Coverslip holder blueprint for 3-D printing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63665/Supplemental File 5.stl" target="_blank">Please click here to download this File.</a>

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.8K Views.  University of New Mexico School of Medicine. Dit SiMPull-protocol maakt een kwantificering van eiwitfosforylering mogelijk door de etiketterings- en fixatieomstandigheden van antilichamen te verbeteren, autofluorescentie in het groene kanaal te verminderen en robuuste analysealgoritmen voor één molecuul te bieden. Het belangrijkste voordeel van SiMPull is dat het ons in staat stelt om de fosforyleringsstatus van individuele intacte eiwitten te ondervragen. Met deze methode kunnen we nieuwe informatie verkrijgen die niet toegankelijk i...

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

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