Universal Non-Antibody Detection of Protein Phosphorylation Using pIMAGO Anton B. Iliuk1,2 and W. Andy Tao1,2,3,4,5 1
Department of Biochemistry, Purdue University, West Lafayette, Indiana Tymora Analytical Operations, LLC, West Lafayette, Indiana 3 Department of Chemistry, Purdue University, West Lafayette, Indiana 4 Department of Medicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, Indiana 5 Corresponding author ([email protected]) 2
This article describes methods for a new, non-antibody phosphorylation detection reagent, termed pIMAGO (phospho-imaging). This novel reagent takes advantage not only of the unique properties of the soluble nanoparticles, but also of the multiple functionalities of the molecule, allowing for highly selective, sensitive, and quantitative assessment of protein phosphorylation without using radioactive isotopes or phospho-specific antibodies. The methods allow for multiplexed detection of phosphorylation and total protein amount simultaneously. The straightforward and routine detection and quantitation of general phosphorylation on any site of any protein can be performed in western blot C 2015 by John Wiley & Sons, Inc. and ELISA formats. Keywords: phosphoprotein detection r phosphorylation r ELISA r western blot r kinase assay r high-throughput screening
How to cite this article: Iliuk, A.B. and Tao, W.A. 2015. Universal Non-Antibody Detection of Protein Phosphorylation Using pIMAGO. Curr. Protoc. Chem. Biol. 7:17-25. doi: 10.1002/9780470559277.ch140208
INTRODUCTION Protein phosphorylation is a crucial post-translational modification that regulates a broad range of cellular activities including the cell cycle, differentiation, metabolism, and signaling (Hunter, 2000; Pawson, 2004). Abnormal phosphorylation events are implicated in many disease states (Blume-Jensen and Hunter, 2001). Therefore, assessing the phosphorylation status of an individual protein or classes of proteins, qualitatively or quantitatively, has become a routine but extremely important task in many laboratories. Common methods for phosphorylation analyses include the use of phospho-specific antibodies, 32 P radioactive labeling, and mass spectrometry. The method of choice may vary depending on the specific question being asked or the availability of specialized equipment or reagents. The drawbacks of the commonly used methods include low sensitivity, poor specificity, irreproducibility, intensive labor, or safety concerns. A simple and reliable assay for routine detection of phosphorylation in typically heterogeneous biological samples is urgently needed to evaluate the dynamics of phosphorylation and provide important insight into how signaling networks function and interact. This article describes methods for non-antibody-based universal detection of protein phosphorylation, termed pIMAGO (phospho-imaging). The technology is based on water-soluble, globular nanopolymers (i.e., dendrimers) that are multi-functionalized with titanium for phosphate recognition and biotin for detection (Iliuk et al., 2011, Current Protocols in Chemical Biology 7:17-25, March 2015 Published online March 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/9780470559277.ch140208 C 2015 John Wiley & Sons, Inc. Copyright
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Figure 1 Graphic illustration (top) and proposed schematic structure (bottom) of the pIMAGO molecule. The structure includes the dendrimer foundation functionalized with phosphonate-bound titanium ions and biotin groups.
Iliuk et al., 2012). A simplified representative illustration of pIMAGO is shown in Figure 1. pIMAGO can be primarily utilized for selective detection of phosphorylated proteins bound to solid-phase formats such as 96-well plates or PVDF membranes. Each pIMAGO molecule contains multiple functional groups, which improves the detection efficiency and allows the analysis of low abundance phosphoproteins in biological samples. The general protocols detailed here enable phosphoprotein detection after immobilization on a membrane (Basic Protocol 1; western blot format) or in a 96-well plate (Basic Protocol 2; Enzyme-Linked ImmunoSorbent Assay—ELISA format). BASIC PROTOCOL 1
Non-Ab Detection of Phosphorylation Using pIMAGO
pIMAGO-BASED PHOSPHOPROTEIN DETECTION IN WESTERN BLOT FORMAT On-membrane detection (i.e., western blotting) using a phospho-specific antibody is the most common method of phosphoprotein detection in the laboratory. Despite this decades-long practice, there are multiple shortcomings that limit its applications. First, the availability of a phospho-specific antibody requires prior knowledge of the phosphorylation site, thus limiting the analysis to well-characterized phosphorylation events. Second, an effective phospho-specific antibody must be made for each phosphosite and phosphoprotein, making assays very expensive. Third, despite improvements in antibody generation methods, phospho-specific antibodies are difficult to develop due to poor selectivity, inconsistent quality, and high cost. Phospho-specific antibodies are extremely valuable for analyzing specific phosphorylation events, but they are sub-optimal for discovery-based experiments in which phosphorylation sites are unknown or highquality antibodies are unavailable. Usually, the first step in molecular signaling studies is to identify whether the phosphorylation status of a protein of interest changes under specific conditions; a positive identification can then be followed by an in-depth examination of modification sites. pIMAGO-based detection is ideal for this type of initial phosphorylation analysis.
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Figure 2 Procedural workflows of pIMAGO-based detection of phosphorylated proteins on (A) membrane or (B) 96-well plate. X = horseradish peroxidase or fluorophore conjugated to avidin. P denotes phosphoprotein.
pIMAGO-based phosphoprotein detection on a membrane is a very simple protocol, similar to a typical western blotting procedure (a workflow is shown in Fig. 2A). The primary difference is that biotin-linked pIMAGO reagent is used instead of a primary antibody, and an avidin-peroxidase or avidin-fluorophore conjugate is used for detection instead of a secondary antibody. Control samples should also be analyzed in parallel to detect any changes in phosphorylation (e.g., in vitro kinase assays with and without ATP).
Materials Phosphorylated protein (e.g., β-casein) as a control 4× SDS sample loading buffer 200 mM dithiothreitol solution (prepare fresh) 400 mM iodoacetamide solution (prepare fresh and protect from light) Blocking buffer for western blot (see recipe) pIMAGO reagent (Tymora Analytical, cat. no. PMGO) pIMAGO buffer (see recipe) Washing buffer (see recipe) Avidin-peroxidase conjugate, for ECL-based detection (Sigma, cat. no. A3151) Avidin-fluorophore conjugate, for fluorescence-based detection (multiple suppliers with various fluorophores are available) 1× TBST (see recipe) Additional reagents and equipment for SDS-PAGE (Gallagher, 2012) and immunoblotting (Gallagher, 2001, Ursitti et al., 2001). Run SDS-PAGE, transfer, and block 1. Boil the substrate of interest, phosphoprotein control (e.g., β-casein), and negative control (e.g., bovine serum albumin, alkaline phosphatase-treated β-casein, or substrate without ATP) samples for 5 min in 1× SDS sample loading buffer supplemented with 20 mM dithiothreitol, and let them cool to room temperature. 2. Add 80 mM (final) iodoacetamide (from a 400 mM stock) directly to the samples, incubate in the dark for 15 min, and then load the samples onto a denaturing
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polyacrylamide gel. Load one well with 10 to 100 ng of the control phosphoprotein. Treating the samples with iodoacetamide is optional, but can improve specificity. Iodoacetamide alkylates the reduced cysteine residues and prevents pIMAGO from binding to them.
3. Fractionate the proteins by SDS-PAGE, and trim the gel to remove the dye front. Removing the dye front from the gel prior to transfer will reduce background in the detection step.
4. Transfer the proteins to a PVDF or nitrocellulose membrane using standard procedure following the manufacturer’s instructions. Any transfer buffer may be used, but Tris-glycine transfer buffer results in lowest background. If fluorescence-based detection will be performed, use a special membrane with low autofluorescence. IMPORTANT NOTE: In many cases, the transfer system itself might contain contaminants, increasing the nonspecific background signal. To reduce this, we strongly recommend including a second piece of PVDF membrane before the gel to bind any of these contaminants (suggested set-up: filter-membrane-gel-membrane-filter). This step is not necessary for nitrocellulose.
5. Block the membrane in blocking buffer (e.g., 10 ml for a mini blot) for 1 hr at room temperature with slight shaking. Blocking can also be carried out overnight at 4°C.
Bind pIMAGO nanoparticles to phosphoproteins 6. Prepare 1:1,000 mixture of pIMAGO reagent in pIMAGO buffer and mix well. For example, dilute 10 μl pIMAGO in 10 ml pIMAGO buffer for a mini gel. 7. Decant the blocking buffer, add the prepared pIMAGO mixture to the membrane, and incubate 1 hr with slight shaking. 8. Wash the membrane 3 times with 10 to 20 ml washing buffer and once with 1× TBST, 5 min each wash.
Bind avidin conjugates 9. Prepare 1:1,000 mixture of avidin-peroxidase or avidin-fluorophore in blocking buffer (e.g., 10 μl avidin-conjugate reagent in 10 ml blocking buffer for a mini gel). 10. Decant the washing buffer, add the diluted avidin conjugate to the membrane, and incubate 1 hr with slight shaking. 11. Wash the membrane 3 times with 1× TBST, 5 min each wash.
Detect and quantify phosphoprotein signals 12. Detect the pIMAGO signal using a fluorescence scanner (for avidin-fluorophore conjugate) or peroxidase chemiluminescence substrate and film (for avidin-peroxidase conjugate). Typically, do not expose the film for more than 1 to 2 min to avoid high background. There is no need to dry the membrane for fluorescence detection.
13. Quantify the signal using band density analysis software for chemiluminescence (e.g., ImageJ) or fluorescence quantitation software typically supplied with fluorescence scanner instruments. Non-Ab Detection of Phosphorylation Using pIMAGO
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pIMAGO-BASED PHOSPHOPROTEIN DETECTION IN ELISA FORMAT As with the on-membrane detection method described in Basic Protocol 1, pIMAGO can be easily adapted for phosphoprotein detection in 96- or 384-well plates (ELISA format). Similarly, this procedure permits general screening to determine whether or not a sample or set of samples is phosphorylated and the extent of phosphorylation. Unlike western blotting, however, microplate-based detection can be accomplished with much higher throughput, generating thousands of data points in one day. As a result, microplate-based detection using pIMAGO is the procedure of choice for high-throughput kinase activity profiling and inhibitor screening.
BASIC PROTOCOL 2
pIMAGO-based phosphoprotein detection in a microplate is similar to a standard ELISA procedure (see workflow in Fig. 2B). After protein immobilization in a plate by passive adsorption, pIMAGO-biotin is bound to phosphoproteins and detected and quantified using avidin-peroxidase or avidin-fluorophore conjugates. Control samples are recommended to quantitatively measure phosphorylation changes (e.g., in vitro kinase assays with and without ATP).
Materials Phosphorylated protein (e.g., β-casein) as a control Carbonate buffer (see recipe) Blocking buffer (see recipe) 1× TBST (see recipe) pIMAGO reagent (Tymora Analytical, cat. no. PMGO) pIMAGO buffer (see recipe) Avidin-peroxidase conjugate, for colorimetry-based detection (Sigma, cat. no. A3151) Avidin-fluorophore conjugate, for fluorescence-based detection (multiple suppliers with various fluorophores are available) Colorimetric TMB peroxidase substrate kit, for colorimetry-based detection (Bio-Rad, cat. no. 172-1066) 2% Oxalic acid prepared in water (protect from light) 96-well, clear High Bind polystyrene plate (Sigma, cat. no. CLS3590) Bind phosphoproteins in a 96-well plate and block 1. Prepare a solution of the substrate of interest, phosphoprotein control (e.g., β-casein), and a negative control sample (e.g., bovine serum albumin, alkaline phosphatase-treated β-casein, or substrate without ATP) in carbonate buffer. If protein amount is known, prepare 10 to 500 ng of the protein (or protein mixture) per 100 μl of carbonate buffer per well.
2. Add 100 μl of each mixture into the appropriate wells of a 96-well clear, High Bind polystyrene plate, and incubate overnight at 4°C, with shaking at 400 to 600 rpm, to bind the proteins to the plate. 3. Remove the solution from each well, add 150 μl the blocking buffer into each well, and incubate 2 to 3 min at room temperature with shaking at 400 to 600 rpm. 4. Remove the blocking buffer from each well, add 150 μl of blocking buffer, and incubate 30 min at room temperature with shaking at 400 to 600 rpm. At this stage, additional manipulations can be carried out (e.g., kinase/phosphatase assay, inhibitor screening). Make sure to wash the wells three times with the 1× TBST after each treatment.
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Bind pIMAGO nanoparticles to phosphoproteins 5. In a fresh tube, prepare a 1:100 mixture of pIMAGO reagent in pIMAGO buffer (1 μl of the reagent for every 100 μl of buffer per well). 6. Remove the blocking buffer from each well, add 100 μl of pIMAGO solution, and incubate 1 hr at room temperature with shaking at 400 to 600 rpm. 7. Remove the pIMAGO solution from each well, add 150 μl pIMAGO buffer to each well, and incubate 2 to 3 min at room temperature with shaking (400 to 600 rpm). 8. Remove the pIMAGO buffer from each well, and repeat step 7 two more times, for a total of three washes with pIMAGO buffer. 9. Remove the pIMAGO buffer from each well, add 150 μl blocking buffer, and incubate 15 min at room temperature with shaking (400 to 600 rpm).
Bind avidin conjugates 10. In a fresh tube, prepare 1:100 mixture of avidin-peroxidase or avidin-fluorophore in blocking buffer (dilute 1 μl avidin conjugate in 100 μl blocking buffer per well). 11. Remove the blocking buffer from each well, add 100 μl avidin-conjugate solution to each well, and incubate the plate 1 hr at room temperature with shaking (400 to 600 rpm). 12. Remove the solution from each well, add 150 μl of 1× TBST to each well, and incubate 2 to 3 min at room temperature with shaking (400 to 600 rpm). 13. Repeat step 12 two more times for a total of three washes with 1× TBST, and empty the wells.
Detect and quantify phosphoprotein signals 14a. If avidin-fluorophore conjugate was used, the fluorescence signal may be detected using a fluorescence scanner. 14b. If avidin-peroxidase conjugate was used, perform colorimetric detection using the TMB peroxidase substrate kit. Prepare 9:1 mixture of the Colorimetric Substrates A and B (prepare fresh each time before detection), add 100 μl to each well, incubate with shaking until the solution turns green (usually 1 to 2 min), and stop the reaction by adding 150 μl of 2% oxalic acid solution. Alternatively, other peroxidase substrates can be used for chromogenic or chemiluminescent detection. However, for chemiluminescence-based detection, a black plate with non-transparent walls must be used.
15. Measure the absorbance at 415 nm in a microplate reader. 16. Quantify signal using absorbance analysis software for chromogenic detection, or fluorescence quantitation software typically supplied with scanning instruments.
REAGENTS AND SOLUTIONS Use Milli-Q water or equivalent in all recipes and protocol steps.
1× Tris-buffered saline with Tween 20 (TBST)
Non-Ab Detection of Phosphorylation Using pIMAGO
50 mM Tris·Cl, pH 7.5 150 mM NaCl Store indefinitely at room temperature Add fresh: 0.1% Tween 20
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Alternatively, 10× TBS can be prepared without Tween 20, and Tween 20 can then be added to each 1× buffer dilution.
Blocking buffer for microplate 1× TBST (see recipe) Add fresh: 1% bovine serum albumin Store up to 1 year at 4°C Blocking buffer for western blot 1× TBST (see recipe) Add fresh: 0.5% bovine serum albumin 0.1% PAMAM generation 4 dendrimer (Sigma, cat. no. 412449) Store up to 1 year at 4°C Carbonate buffer 30 mM sodium carbonate 70 mM sodium bicarbonate The pH should be 9.2 to 9.6 Store up to 1 year at 4°C pIMAGO buffer 1% trifluoroacetic acid 500 mM glycolic acid Store indefinitely at room temperature Washing buffer 0.1% trifluoroacetic acid 50 mM 2,5-dihydrobenzoic acid Store in a dark bottle indefinitely at room temperature COMMENTARY Background Information Titanium ion or titanium dioxide has strong affinity for phosphorylated residues (Nawrocki et al., 2004; Torta et al., 2009). Although binding is mainly based on chargecharge interactions, in the presence of benzoic or α-hydroxy acids, titanium shows remarkable specificity toward phosphate groups over other negatively charged molecules (Larsen et al., 2005; Jensen and Larsen, 2007). We have previously demonstrated the highly effective utility of titanium-based enrichment of phosphopeptides for mass spectrometry analysis under homogeneous conditions (PolyMAC technology; Iliuk et al., 2010). For this application, we utilized a soluble polyamidoamine synthetic nanopolymer (e.g., dendrimer) whose hyperbranched surface can be functionalized with desired chemical groups (i.e., titanium for phosphate binding). The advantages of using dendrimers include high solubility, high struc-
tural and chemical homogeneity, compact spherical shape, and controlled surface functionalities (Boas and Heegaard, 2004). The homogeneous and hyper-branched nature of titanium-functionalized nanopolymers further enhances specificity toward phosphorylated molecules. Following the initial mass spectrometrybased applications, we expanded the concept of titanium-functionalized nanopolymers to develop a universal phosphoprotein detection approach, which we termed pIMAGO. Each dendrimer molecule was synthesized to contain titanium metal ions for highly specific binding to phosphoproteins and biotin groups for subsequent signal detection. pIMAGO bound to proteins immobilized on a membrane or a plate can be detected with horseradish peroxidase (HRP) conjugated to avidin. Under the strong acidic conditions provided by the pIMAGO buffer, the titanium-functionalized nanopolymer is capable of binding to
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Table 1 Troubleshooting pIMAGO-based detection
Problem
Possible Cause
Solution
High background on membrane
Contaminants in the transfer system
Include a second piece of PVDF membrane in front of the gel during transfer to adsorb contaminants
Too much signal/background
Excess avidin conjugate
Reduce the amount of avidin conjugate (e.g., peroxidase)
Too much signal/background on membrane
Exposure time is too long
Reduce the exposure time to 30 sec
Potential false positives
Control is lacking
Include negative controls by either excluding ATP during the kinase reaction or dephosphorylating the sample with a general phosphatase
No detectable signal
Procedural problems or no phosphoprotein present
Include a known phosphoprotein (e.g., β-casein) as a positive control
phosphorylated groups irrespective of the surrounding amino acid sequence or different phosphosites. Moreover, because each nanopolymer is functionalized with multiple biotin groups, the signal from each binding event is amplified, thus allowing the detection of low abundance phosphoproteins. Using this method, the phosphorylation levels of proteins of interest under physiological conditions can be readily detected in western blot- or ELISAlike procedures, without the need for radiolabeled ATP or phospho-specific antibodies.
Critical Parameters
Non-Ab Detection of Phosphorylation Using pIMAGO
Choice of detection method Chemiluminescent and colorimetric substrates are the most common and convenient methods for western blot and ELISA, respectively. Although peroxidase-based detection using chemiluminescence for membrane and chromogenic substrate for microplate-based analysis are described here, signal detection is not limited to these. Any detection method can be used if avidin or streptavidin is conjugated to the signaling molecule, including peroxidase, alkaline phosphatase, dyes, or any wavelength fluorophore. In fact, we recommend avidin-fluorophore conjugates, because they provide a consistent signal that is independent of enzyme activity or substrate depletion, thereby resulting in more accurate signal quantitation. The protocols, as described, are suitable for any signaling molecule and need only be appropriately modified at the signal detection step.
Selection of binding platform For on-membrane phosphoprotein detection, it is necessary to ensure that a highquality PVDF membrane is used to avoid potentially high background. If fluorescencebased detection is desirable, a specialty membrane with low autofluorescence should be selected. Similar properties should be kept in mind for on-plate detection as well, where a low-autofluorescence plate should be used for fluorophore-based detection. In addition, if a chemiluminescent substrate will be used for on-plate detection, a black non-translucent plate needs to be used to avoid signal crossover between wells.
Troubleshooting Table 1 describes problems commonly encountered with the protocols described in this article, along with possible causes and suggestions for overcoming or avoiding these problems.
Anticipated Results Basic Protocols 1 and 2 are designed to be simple and routine procedures for detection of general protein phosphorylation. We expect that any phosphoprotein sample of over 1 ng will be easily detected by either avidin-conjugated detection method. Because pIMAGO binds to any phosphosite, however, one cannot differentiate which sites on a protein are phosphorylated. In addition, even after a stimulus, a change in phosphorylation may not be observed if it affects only one minor site but not the overall phosphorylation level of a
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protein of interest. Therefore, this approach should be used as a method to screen for phosphorylation changes, and combined with other approaches (e.g., mass spectrometry) for indepth analysis of targeted phosphoproteins.
Time Consideration After protein immobilization on a membrane (protein transfer) or in a microplate, the pIMAGO detection procedure will take less than 4 hr. If the procedure cannot be completed on the same day, membranes or microplates may be left to block overnight at 4°C, prior to pIMAGO binding step.
Conflict of Interest pIMAGO is a commercial product marketed by Tymora Analytical, of which the coauthors are founders.
Acknowledgements This project was funded in part by NSF CAREER award CHE-0645020, National Institutes of Health grants 1R01GM088317 and R43EB15809-1, and NSF grant IIP-1256600.
Literature Cited Blume-Jensen, P. and Hunter, T. 2001. Oncogenic kinase signalling. Nature 411:355-365. Boas, U. and Heegaard, P. M. 2004. Dendrimers in drug research. Chem. Soc. Rev. 33:43-63. Gallagher, S. 2001. Immunoblot detection. Curr. Protoc. Prot. Sci. 4:10.10.1-10.10.12. Gallagher, S.R. 2012. One-dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Prot. Sci. 68:10.1.1-10.1.44. Hunter, T. 2000. Signaling–2000 and beyond. Cell 100:113-127.
Iliuk, A., Martinez, J.S., Hall, M.C., and Tao, W.A. 2011. Phosphorylation assay based on multifunctionalized soluble nanopolymer. Anal. Chem. 83:2767-2774. Iliuk, A.B., Martin, V.A., Alicie, B.M., Geahlen, R.L., and Tao, W.A. 2010. In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion-functionalized soluble nanopolymers. Mol. Cell Proteomics. 9:2162-2172. Iliuk, A., Liu, X.S., Xue, L., Liu, X., and Tao, W.A. 2012. Chemical visualization of phosphoproteomes on membrane. Mol. Cell. Proteomics 11:629-639. Jensen, S.S. and Larsen, M.R. 2007. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid. Commun. Mass Spectrom. 21:3635-3645. Larsen, M.R., Thingholm, T.E., Jensen, O.N., Roepstorff, P., and Jorgensen, T.J. 2005. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell Proteomics. 4:873-886. Nawrocki, J., Dunlap, C., McCormick, A., and Carr, P.W. 2004. Part I. Chromatography using ultrastable metal oxide-based stationary phases for HPLC. J. Chromato. A 1028:1-30. Pawson, T. 2004. Specificity in signal transduction: From phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 116:191-203. Torta, F., Fusi, M., Casari, C.S., Bottani, C.E., and Bachi, A. 2009. Titanium dioxide coated MALDI plate for on target analysis of phosphopeptides. J. Proteome. Res. 8:19321942. Ursitti, J.A., Mozdzanowski, J., and Speicher, D.W. 2001. Electroblotting from polyacrylamide gels. Curr. Protoc. Prot. Sci. 00:10.7.1-10.7.14.
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Department of Biochemistry, Purdue University, West Lafayette, Indiana Tymora Analytical Operations, LLC, West Lafayette, Indiana 3 Department of Chemistry, Purdue University, West Lafayette, Indiana 4 Department of Medicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, Indiana 5 Corresponding author ([email protected]) 2
This article describes methods for a new, non-antibody phosphorylation detection reagent, termed pIMAGO (phospho-imaging). This novel reagent takes advantage not only of the unique properties of the soluble nanoparticles, but also of the multiple functionalities of the molecule, allowing for highly selective, sensitive, and quantitative assessment of protein phosphorylation without using radioactive isotopes or phospho-specific antibodies. The methods allow for multiplexed detection of phosphorylation and total protein amount simultaneously. The straightforward and routine detection and quantitation of general phosphorylation on any site of any protein can be performed in western blot C 2015 by John Wiley & Sons, Inc. and ELISA formats. Keywords: phosphoprotein detection r phosphorylation r ELISA r western blot r kinase assay r high-throughput screening
How to cite this article: Iliuk, A.B. and Tao, W.A. 2015. Universal Non-Antibody Detection of Protein Phosphorylation Using pIMAGO. Curr. Protoc. Chem. Biol. 7:17-25. doi: 10.1002/9780470559277.ch140208
INTRODUCTION Protein phosphorylation is a crucial post-translational modification that regulates a broad range of cellular activities including the cell cycle, differentiation, metabolism, and signaling (Hunter, 2000; Pawson, 2004). Abnormal phosphorylation events are implicated in many disease states (Blume-Jensen and Hunter, 2001). Therefore, assessing the phosphorylation status of an individual protein or classes of proteins, qualitatively or quantitatively, has become a routine but extremely important task in many laboratories. Common methods for phosphorylation analyses include the use of phospho-specific antibodies, 32 P radioactive labeling, and mass spectrometry. The method of choice may vary depending on the specific question being asked or the availability of specialized equipment or reagents. The drawbacks of the commonly used methods include low sensitivity, poor specificity, irreproducibility, intensive labor, or safety concerns. A simple and reliable assay for routine detection of phosphorylation in typically heterogeneous biological samples is urgently needed to evaluate the dynamics of phosphorylation and provide important insight into how signaling networks function and interact. This article describes methods for non-antibody-based universal detection of protein phosphorylation, termed pIMAGO (phospho-imaging). The technology is based on water-soluble, globular nanopolymers (i.e., dendrimers) that are multi-functionalized with titanium for phosphate recognition and biotin for detection (Iliuk et al., 2011, Current Protocols in Chemical Biology 7:17-25, March 2015 Published online March 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/9780470559277.ch140208 C 2015 John Wiley & Sons, Inc. Copyright
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Figure 1 Graphic illustration (top) and proposed schematic structure (bottom) of the pIMAGO molecule. The structure includes the dendrimer foundation functionalized with phosphonate-bound titanium ions and biotin groups.
Iliuk et al., 2012). A simplified representative illustration of pIMAGO is shown in Figure 1. pIMAGO can be primarily utilized for selective detection of phosphorylated proteins bound to solid-phase formats such as 96-well plates or PVDF membranes. Each pIMAGO molecule contains multiple functional groups, which improves the detection efficiency and allows the analysis of low abundance phosphoproteins in biological samples. The general protocols detailed here enable phosphoprotein detection after immobilization on a membrane (Basic Protocol 1; western blot format) or in a 96-well plate (Basic Protocol 2; Enzyme-Linked ImmunoSorbent Assay—ELISA format). BASIC PROTOCOL 1
Non-Ab Detection of Phosphorylation Using pIMAGO
pIMAGO-BASED PHOSPHOPROTEIN DETECTION IN WESTERN BLOT FORMAT On-membrane detection (i.e., western blotting) using a phospho-specific antibody is the most common method of phosphoprotein detection in the laboratory. Despite this decades-long practice, there are multiple shortcomings that limit its applications. First, the availability of a phospho-specific antibody requires prior knowledge of the phosphorylation site, thus limiting the analysis to well-characterized phosphorylation events. Second, an effective phospho-specific antibody must be made for each phosphosite and phosphoprotein, making assays very expensive. Third, despite improvements in antibody generation methods, phospho-specific antibodies are difficult to develop due to poor selectivity, inconsistent quality, and high cost. Phospho-specific antibodies are extremely valuable for analyzing specific phosphorylation events, but they are sub-optimal for discovery-based experiments in which phosphorylation sites are unknown or highquality antibodies are unavailable. Usually, the first step in molecular signaling studies is to identify whether the phosphorylation status of a protein of interest changes under specific conditions; a positive identification can then be followed by an in-depth examination of modification sites. pIMAGO-based detection is ideal for this type of initial phosphorylation analysis.
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Figure 2 Procedural workflows of pIMAGO-based detection of phosphorylated proteins on (A) membrane or (B) 96-well plate. X = horseradish peroxidase or fluorophore conjugated to avidin. P denotes phosphoprotein.
pIMAGO-based phosphoprotein detection on a membrane is a very simple protocol, similar to a typical western blotting procedure (a workflow is shown in Fig. 2A). The primary difference is that biotin-linked pIMAGO reagent is used instead of a primary antibody, and an avidin-peroxidase or avidin-fluorophore conjugate is used for detection instead of a secondary antibody. Control samples should also be analyzed in parallel to detect any changes in phosphorylation (e.g., in vitro kinase assays with and without ATP).
Materials Phosphorylated protein (e.g., β-casein) as a control 4× SDS sample loading buffer 200 mM dithiothreitol solution (prepare fresh) 400 mM iodoacetamide solution (prepare fresh and protect from light) Blocking buffer for western blot (see recipe) pIMAGO reagent (Tymora Analytical, cat. no. PMGO) pIMAGO buffer (see recipe) Washing buffer (see recipe) Avidin-peroxidase conjugate, for ECL-based detection (Sigma, cat. no. A3151) Avidin-fluorophore conjugate, for fluorescence-based detection (multiple suppliers with various fluorophores are available) 1× TBST (see recipe) Additional reagents and equipment for SDS-PAGE (Gallagher, 2012) and immunoblotting (Gallagher, 2001, Ursitti et al., 2001). Run SDS-PAGE, transfer, and block 1. Boil the substrate of interest, phosphoprotein control (e.g., β-casein), and negative control (e.g., bovine serum albumin, alkaline phosphatase-treated β-casein, or substrate without ATP) samples for 5 min in 1× SDS sample loading buffer supplemented with 20 mM dithiothreitol, and let them cool to room temperature. 2. Add 80 mM (final) iodoacetamide (from a 400 mM stock) directly to the samples, incubate in the dark for 15 min, and then load the samples onto a denaturing
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polyacrylamide gel. Load one well with 10 to 100 ng of the control phosphoprotein. Treating the samples with iodoacetamide is optional, but can improve specificity. Iodoacetamide alkylates the reduced cysteine residues and prevents pIMAGO from binding to them.
3. Fractionate the proteins by SDS-PAGE, and trim the gel to remove the dye front. Removing the dye front from the gel prior to transfer will reduce background in the detection step.
4. Transfer the proteins to a PVDF or nitrocellulose membrane using standard procedure following the manufacturer’s instructions. Any transfer buffer may be used, but Tris-glycine transfer buffer results in lowest background. If fluorescence-based detection will be performed, use a special membrane with low autofluorescence. IMPORTANT NOTE: In many cases, the transfer system itself might contain contaminants, increasing the nonspecific background signal. To reduce this, we strongly recommend including a second piece of PVDF membrane before the gel to bind any of these contaminants (suggested set-up: filter-membrane-gel-membrane-filter). This step is not necessary for nitrocellulose.
5. Block the membrane in blocking buffer (e.g., 10 ml for a mini blot) for 1 hr at room temperature with slight shaking. Blocking can also be carried out overnight at 4°C.
Bind pIMAGO nanoparticles to phosphoproteins 6. Prepare 1:1,000 mixture of pIMAGO reagent in pIMAGO buffer and mix well. For example, dilute 10 μl pIMAGO in 10 ml pIMAGO buffer for a mini gel. 7. Decant the blocking buffer, add the prepared pIMAGO mixture to the membrane, and incubate 1 hr with slight shaking. 8. Wash the membrane 3 times with 10 to 20 ml washing buffer and once with 1× TBST, 5 min each wash.
Bind avidin conjugates 9. Prepare 1:1,000 mixture of avidin-peroxidase or avidin-fluorophore in blocking buffer (e.g., 10 μl avidin-conjugate reagent in 10 ml blocking buffer for a mini gel). 10. Decant the washing buffer, add the diluted avidin conjugate to the membrane, and incubate 1 hr with slight shaking. 11. Wash the membrane 3 times with 1× TBST, 5 min each wash.
Detect and quantify phosphoprotein signals 12. Detect the pIMAGO signal using a fluorescence scanner (for avidin-fluorophore conjugate) or peroxidase chemiluminescence substrate and film (for avidin-peroxidase conjugate). Typically, do not expose the film for more than 1 to 2 min to avoid high background. There is no need to dry the membrane for fluorescence detection.
13. Quantify the signal using band density analysis software for chemiluminescence (e.g., ImageJ) or fluorescence quantitation software typically supplied with fluorescence scanner instruments. Non-Ab Detection of Phosphorylation Using pIMAGO
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pIMAGO-BASED PHOSPHOPROTEIN DETECTION IN ELISA FORMAT As with the on-membrane detection method described in Basic Protocol 1, pIMAGO can be easily adapted for phosphoprotein detection in 96- or 384-well plates (ELISA format). Similarly, this procedure permits general screening to determine whether or not a sample or set of samples is phosphorylated and the extent of phosphorylation. Unlike western blotting, however, microplate-based detection can be accomplished with much higher throughput, generating thousands of data points in one day. As a result, microplate-based detection using pIMAGO is the procedure of choice for high-throughput kinase activity profiling and inhibitor screening.
BASIC PROTOCOL 2
pIMAGO-based phosphoprotein detection in a microplate is similar to a standard ELISA procedure (see workflow in Fig. 2B). After protein immobilization in a plate by passive adsorption, pIMAGO-biotin is bound to phosphoproteins and detected and quantified using avidin-peroxidase or avidin-fluorophore conjugates. Control samples are recommended to quantitatively measure phosphorylation changes (e.g., in vitro kinase assays with and without ATP).
Materials Phosphorylated protein (e.g., β-casein) as a control Carbonate buffer (see recipe) Blocking buffer (see recipe) 1× TBST (see recipe) pIMAGO reagent (Tymora Analytical, cat. no. PMGO) pIMAGO buffer (see recipe) Avidin-peroxidase conjugate, for colorimetry-based detection (Sigma, cat. no. A3151) Avidin-fluorophore conjugate, for fluorescence-based detection (multiple suppliers with various fluorophores are available) Colorimetric TMB peroxidase substrate kit, for colorimetry-based detection (Bio-Rad, cat. no. 172-1066) 2% Oxalic acid prepared in water (protect from light) 96-well, clear High Bind polystyrene plate (Sigma, cat. no. CLS3590) Bind phosphoproteins in a 96-well plate and block 1. Prepare a solution of the substrate of interest, phosphoprotein control (e.g., β-casein), and a negative control sample (e.g., bovine serum albumin, alkaline phosphatase-treated β-casein, or substrate without ATP) in carbonate buffer. If protein amount is known, prepare 10 to 500 ng of the protein (or protein mixture) per 100 μl of carbonate buffer per well.
2. Add 100 μl of each mixture into the appropriate wells of a 96-well clear, High Bind polystyrene plate, and incubate overnight at 4°C, with shaking at 400 to 600 rpm, to bind the proteins to the plate. 3. Remove the solution from each well, add 150 μl the blocking buffer into each well, and incubate 2 to 3 min at room temperature with shaking at 400 to 600 rpm. 4. Remove the blocking buffer from each well, add 150 μl of blocking buffer, and incubate 30 min at room temperature with shaking at 400 to 600 rpm. At this stage, additional manipulations can be carried out (e.g., kinase/phosphatase assay, inhibitor screening). Make sure to wash the wells three times with the 1× TBST after each treatment.
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Bind pIMAGO nanoparticles to phosphoproteins 5. In a fresh tube, prepare a 1:100 mixture of pIMAGO reagent in pIMAGO buffer (1 μl of the reagent for every 100 μl of buffer per well). 6. Remove the blocking buffer from each well, add 100 μl of pIMAGO solution, and incubate 1 hr at room temperature with shaking at 400 to 600 rpm. 7. Remove the pIMAGO solution from each well, add 150 μl pIMAGO buffer to each well, and incubate 2 to 3 min at room temperature with shaking (400 to 600 rpm). 8. Remove the pIMAGO buffer from each well, and repeat step 7 two more times, for a total of three washes with pIMAGO buffer. 9. Remove the pIMAGO buffer from each well, add 150 μl blocking buffer, and incubate 15 min at room temperature with shaking (400 to 600 rpm).
Bind avidin conjugates 10. In a fresh tube, prepare 1:100 mixture of avidin-peroxidase or avidin-fluorophore in blocking buffer (dilute 1 μl avidin conjugate in 100 μl blocking buffer per well). 11. Remove the blocking buffer from each well, add 100 μl avidin-conjugate solution to each well, and incubate the plate 1 hr at room temperature with shaking (400 to 600 rpm). 12. Remove the solution from each well, add 150 μl of 1× TBST to each well, and incubate 2 to 3 min at room temperature with shaking (400 to 600 rpm). 13. Repeat step 12 two more times for a total of three washes with 1× TBST, and empty the wells.
Detect and quantify phosphoprotein signals 14a. If avidin-fluorophore conjugate was used, the fluorescence signal may be detected using a fluorescence scanner. 14b. If avidin-peroxidase conjugate was used, perform colorimetric detection using the TMB peroxidase substrate kit. Prepare 9:1 mixture of the Colorimetric Substrates A and B (prepare fresh each time before detection), add 100 μl to each well, incubate with shaking until the solution turns green (usually 1 to 2 min), and stop the reaction by adding 150 μl of 2% oxalic acid solution. Alternatively, other peroxidase substrates can be used for chromogenic or chemiluminescent detection. However, for chemiluminescence-based detection, a black plate with non-transparent walls must be used.
15. Measure the absorbance at 415 nm in a microplate reader. 16. Quantify signal using absorbance analysis software for chromogenic detection, or fluorescence quantitation software typically supplied with scanning instruments.
REAGENTS AND SOLUTIONS Use Milli-Q water or equivalent in all recipes and protocol steps.
1× Tris-buffered saline with Tween 20 (TBST)
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50 mM Tris·Cl, pH 7.5 150 mM NaCl Store indefinitely at room temperature Add fresh: 0.1% Tween 20
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Alternatively, 10× TBS can be prepared without Tween 20, and Tween 20 can then be added to each 1× buffer dilution.
Blocking buffer for microplate 1× TBST (see recipe) Add fresh: 1% bovine serum albumin Store up to 1 year at 4°C Blocking buffer for western blot 1× TBST (see recipe) Add fresh: 0.5% bovine serum albumin 0.1% PAMAM generation 4 dendrimer (Sigma, cat. no. 412449) Store up to 1 year at 4°C Carbonate buffer 30 mM sodium carbonate 70 mM sodium bicarbonate The pH should be 9.2 to 9.6 Store up to 1 year at 4°C pIMAGO buffer 1% trifluoroacetic acid 500 mM glycolic acid Store indefinitely at room temperature Washing buffer 0.1% trifluoroacetic acid 50 mM 2,5-dihydrobenzoic acid Store in a dark bottle indefinitely at room temperature COMMENTARY Background Information Titanium ion or titanium dioxide has strong affinity for phosphorylated residues (Nawrocki et al., 2004; Torta et al., 2009). Although binding is mainly based on chargecharge interactions, in the presence of benzoic or α-hydroxy acids, titanium shows remarkable specificity toward phosphate groups over other negatively charged molecules (Larsen et al., 2005; Jensen and Larsen, 2007). We have previously demonstrated the highly effective utility of titanium-based enrichment of phosphopeptides for mass spectrometry analysis under homogeneous conditions (PolyMAC technology; Iliuk et al., 2010). For this application, we utilized a soluble polyamidoamine synthetic nanopolymer (e.g., dendrimer) whose hyperbranched surface can be functionalized with desired chemical groups (i.e., titanium for phosphate binding). The advantages of using dendrimers include high solubility, high struc-
tural and chemical homogeneity, compact spherical shape, and controlled surface functionalities (Boas and Heegaard, 2004). The homogeneous and hyper-branched nature of titanium-functionalized nanopolymers further enhances specificity toward phosphorylated molecules. Following the initial mass spectrometrybased applications, we expanded the concept of titanium-functionalized nanopolymers to develop a universal phosphoprotein detection approach, which we termed pIMAGO. Each dendrimer molecule was synthesized to contain titanium metal ions for highly specific binding to phosphoproteins and biotin groups for subsequent signal detection. pIMAGO bound to proteins immobilized on a membrane or a plate can be detected with horseradish peroxidase (HRP) conjugated to avidin. Under the strong acidic conditions provided by the pIMAGO buffer, the titanium-functionalized nanopolymer is capable of binding to
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Table 1 Troubleshooting pIMAGO-based detection
Problem
Possible Cause
Solution
High background on membrane
Contaminants in the transfer system
Include a second piece of PVDF membrane in front of the gel during transfer to adsorb contaminants
Too much signal/background
Excess avidin conjugate
Reduce the amount of avidin conjugate (e.g., peroxidase)
Too much signal/background on membrane
Exposure time is too long
Reduce the exposure time to 30 sec
Potential false positives
Control is lacking
Include negative controls by either excluding ATP during the kinase reaction or dephosphorylating the sample with a general phosphatase
No detectable signal
Procedural problems or no phosphoprotein present
Include a known phosphoprotein (e.g., β-casein) as a positive control
phosphorylated groups irrespective of the surrounding amino acid sequence or different phosphosites. Moreover, because each nanopolymer is functionalized with multiple biotin groups, the signal from each binding event is amplified, thus allowing the detection of low abundance phosphoproteins. Using this method, the phosphorylation levels of proteins of interest under physiological conditions can be readily detected in western blot- or ELISAlike procedures, without the need for radiolabeled ATP or phospho-specific antibodies.
Critical Parameters
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Choice of detection method Chemiluminescent and colorimetric substrates are the most common and convenient methods for western blot and ELISA, respectively. Although peroxidase-based detection using chemiluminescence for membrane and chromogenic substrate for microplate-based analysis are described here, signal detection is not limited to these. Any detection method can be used if avidin or streptavidin is conjugated to the signaling molecule, including peroxidase, alkaline phosphatase, dyes, or any wavelength fluorophore. In fact, we recommend avidin-fluorophore conjugates, because they provide a consistent signal that is independent of enzyme activity or substrate depletion, thereby resulting in more accurate signal quantitation. The protocols, as described, are suitable for any signaling molecule and need only be appropriately modified at the signal detection step.
Selection of binding platform For on-membrane phosphoprotein detection, it is necessary to ensure that a highquality PVDF membrane is used to avoid potentially high background. If fluorescencebased detection is desirable, a specialty membrane with low autofluorescence should be selected. Similar properties should be kept in mind for on-plate detection as well, where a low-autofluorescence plate should be used for fluorophore-based detection. In addition, if a chemiluminescent substrate will be used for on-plate detection, a black non-translucent plate needs to be used to avoid signal crossover between wells.
Troubleshooting Table 1 describes problems commonly encountered with the protocols described in this article, along with possible causes and suggestions for overcoming or avoiding these problems.
Anticipated Results Basic Protocols 1 and 2 are designed to be simple and routine procedures for detection of general protein phosphorylation. We expect that any phosphoprotein sample of over 1 ng will be easily detected by either avidin-conjugated detection method. Because pIMAGO binds to any phosphosite, however, one cannot differentiate which sites on a protein are phosphorylated. In addition, even after a stimulus, a change in phosphorylation may not be observed if it affects only one minor site but not the overall phosphorylation level of a
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protein of interest. Therefore, this approach should be used as a method to screen for phosphorylation changes, and combined with other approaches (e.g., mass spectrometry) for indepth analysis of targeted phosphoproteins.
Time Consideration After protein immobilization on a membrane (protein transfer) or in a microplate, the pIMAGO detection procedure will take less than 4 hr. If the procedure cannot be completed on the same day, membranes or microplates may be left to block overnight at 4°C, prior to pIMAGO binding step.
Conflict of Interest pIMAGO is a commercial product marketed by Tymora Analytical, of which the coauthors are founders.
Acknowledgements This project was funded in part by NSF CAREER award CHE-0645020, National Institutes of Health grants 1R01GM088317 and R43EB15809-1, and NSF grant IIP-1256600.
Literature Cited Blume-Jensen, P. and Hunter, T. 2001. Oncogenic kinase signalling. Nature 411:355-365. Boas, U. and Heegaard, P. M. 2004. Dendrimers in drug research. Chem. Soc. Rev. 33:43-63. Gallagher, S. 2001. Immunoblot detection. Curr. Protoc. Prot. Sci. 4:10.10.1-10.10.12. Gallagher, S.R. 2012. One-dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Prot. Sci. 68:10.1.1-10.1.44. Hunter, T. 2000. Signaling–2000 and beyond. Cell 100:113-127.
Iliuk, A., Martinez, J.S., Hall, M.C., and Tao, W.A. 2011. Phosphorylation assay based on multifunctionalized soluble nanopolymer. Anal. Chem. 83:2767-2774. Iliuk, A.B., Martin, V.A., Alicie, B.M., Geahlen, R.L., and Tao, W.A. 2010. In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion-functionalized soluble nanopolymers. Mol. Cell Proteomics. 9:2162-2172. Iliuk, A., Liu, X.S., Xue, L., Liu, X., and Tao, W.A. 2012. Chemical visualization of phosphoproteomes on membrane. Mol. Cell. Proteomics 11:629-639. Jensen, S.S. and Larsen, M.R. 2007. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid. Commun. Mass Spectrom. 21:3635-3645. Larsen, M.R., Thingholm, T.E., Jensen, O.N., Roepstorff, P., and Jorgensen, T.J. 2005. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell Proteomics. 4:873-886. Nawrocki, J., Dunlap, C., McCormick, A., and Carr, P.W. 2004. Part I. Chromatography using ultrastable metal oxide-based stationary phases for HPLC. J. Chromato. A 1028:1-30. Pawson, T. 2004. Specificity in signal transduction: From phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 116:191-203. Torta, F., Fusi, M., Casari, C.S., Bottani, C.E., and Bachi, A. 2009. Titanium dioxide coated MALDI plate for on target analysis of phosphopeptides. J. Proteome. Res. 8:19321942. Ursitti, J.A., Mozdzanowski, J., and Speicher, D.W. 2001. Electroblotting from polyacrylamide gels. Curr. Protoc. Prot. Sci. 00:10.7.1-10.7.14.
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