METHODS FOR DETECTING PROTEIN PHOSPHORYLATION

Introduction

Box 1. R&D Systems DuoSet IC IP-Kinase Assays offer a highly specific and sensitive way to measure endogenous kinase activity. These assays are simple to perform and provide rapid, quantitative results. Each kit provides a kinase-specific antibody conjugated to agarose beads, HRP-conjugated detection reagent, Streptavidin- coated microplate, biotinylated kinase substrate, biotinylated phospho-peptide control, and HRP substrate solution. The captured kinase phosphorylates the provided substrate, and phosphorylation is assessed using colorimetric detection.

Chk1 Activity in HeLa Cells. Kinase activity of endogenous Chk1 in HeLa cell lysates was assayed in the presence of increasing concentrations of SB218078, a specific Chk1 inhibitor, using the Active Chk1 Kinase DuoSet IC IP-Kinase Assay (Catalog # DYC1630). The presence of similar levels of Chk1 protein in each immunoprecipitate was detected by Western blot using an anti-human Chk1 polyclonal antibody (Catalog # AF1630; inset).

Protein kinases transfer phosphate groups from ATP to serine, threonine, or tyrosine residues on protein peptide substrates, directly affecting the activity and function of the target. Radiolabel studies suggest that approximately 30% of proteins in eukaryotic cells are subject to phosphorylation.^1,2 This crucial post-translational modification regulates a broad range of cellular activities including the cell cycle, differentiation, metabolism, and neuronal communication. In addition, abnormal phosphorylation events are implicated in many disease states. When assessing phosphorylation, the method of choice may vary depending on many factors including the specific question being asked and availability of specialized equipment or reagents. This review provides a brief description of several methodologies currently used and addresses some of the benefits and drawbacks associated with each.

Kinase Activity Assays

Protein kinases are often common elements in multiple signaling networks influencing numerous downstream effectors responsible for a biological response. Thus, assessing the activity of a single specific kinase may provide valuable information on parallel pathways.³ Kinase activity within a biological sample is commonly measured in vitro by incubating the immunoprecipitated kinase with an exogenous substrate in the presence of ATP. Measurement of the phosphorylated substrate can be assessed by several reporter systems including colorimetric (Box 1), radioactive, or fluorometric detection.⁴ Although information can be obtained regarding the actions of a specific kinase, assessing enzyme activity in cellular extracts only provides a glimpse of the signaling landscape. Little is revealed about the proteins being modified, and in vitro activity assays do not address the role of potential endogenous phosphatase activity. Direct detection of phosphorylated proteins can provide a more detailed analysis of the cellular response to an external stimulus, as identification of a phosphopeptide provides information regarding the expression and the functional state of that protein.

Phospho-Specific Antibody Development

A classical method of directly measuring protein phosphorylation involves the incubation of whole cells with radiolabeled ³²P-orthophosphate, the generation of cellular extracts, separation of proteins by SDS-PAGE, and exposure to film.^2,5 This labor-intensive method requires many multi-hour incubations and the use of radioisotopes. Other traditional methods include 2-dimensional gel electrophoresis, a technique that assumes phosphorylation will alter the mobility and isoelectric point of the protein.

In light of these laborious methods, the development of phosphorylation-dependent antibodies was a welcome event for researchers. In 1981, the first documented phospho-antibody was produced in rabbits immunized with benzonyl phosphonate conjugated to keyhole limpet hemocyanin (KLH). This antibody broadly recognized proteins containing phosphotyrosine.⁶ Ten years later, phosphorlyation state-specific (phospho-specific) antibodies were developed by immunizing rabbits with synthetic phosphopeptides representing the amino acid sequence surrounding the phosphorylation site of the target protein.⁷ The immune sera was applied to a peptide affinity column to generate a highly specific immunoreagent. The availability of phospho-specific antibodies (Table 1) has opened the door for the improvement of traditional methods as well as the development of new immunoassay techniques. The main caveat in utilizing phospho-specific antibodies in any technique is that successful detection is dependent on the specificity and affinity of the antibody for the phospho-protein of interest.

Table 1. R&D Systems offers a wide range of phospho-specific antibodies that have been validated for multiple applications.

Western Blot

Figure 1. Phosphorylated p53 in CEM Cells. Human T lymphoblast CEM cells were exposed to UV-C light. Cellular extracts generated at 30 or 60 minutes post-irradiation were assessed by Western blot using rabbit anti-human phospho-p53 (S15) polyclonal antibody (Catalog # AF1043, upper panel) or goat anti-human p53 polyclonal antibody (Catalog # AF1355, lower panel). Indicated samples were treated with lambda-phosphatase (lambda-PPase).

The Western blot is the most common method used for assessing the phosphorylation state of a protein, and most cell biology laboratories possess the equipment necessary to perform these experiments. Following separation of the biological sample with SDS- PAGE and subsequent transfer to a membrane (usually PVDF or nitrocellulose), a phospho-specific antibody can be used to identify the protein of interest. The typical Western blot protocol eliminates the hazards and waste disposal requirements associated with the use of radioisotopes. Many phospho-specific antibodies are quite sensitive and can readily detect the phosphorylated protein in a routine sample (e.g., 10-30 µg whole cell extract). Because the measured levels of a phospho-protein may change with treatment or through gel loading errors, researchers often utilize an antibody that detects the total level of the cognate protein (regardless of phosphorylation state) to determine the phosphorylated fraction relative to the total fraction and to serve as an internal loading control (Figure 1). Both chemiluminescent and colorimetric detection methods are common, and molecular weight markers are also generally used to provide information about protein mass. A detailed Western blot protocol can be found at www.RnDSystems.com/go/WesternBlotProtocol.

Enzyme-Linked Immunosorbent Assay (ELISA)

The ELISA has become a powerful method for measuring protein phosphorylation. ELISAs are more quantitative than Western blotting and show great utility in studies that modulate kinase activity and function. The format for this microplate-based assay typically utilizes a capture antibody specific for the desired protein, independent of the phosphorylation state. The target protein, either purified or as a component in a complex heterogeneous sample such as a cell lysate, is then bound to the antibody-coated plate. A detection antibody specific for the phosphorylation site to be analyzed is then added. These assays are typically designed using colorimetric or fluorometric detection. The intensity of the resulting signal is directly proportional to the concentration of phosphorylated protein present in the original sample (Box 2). The phospho-specific ELISA technique confers several advantages over more traditional immunoblotting in the measurement of protein phosphorylation. First, results are easily quantifiable by utilizing a calibrated standard. Second, high specificity is possible due to the use of two antibodies specific for the target protein employed together in the sandwich format. Finally, the higher sensitivity often accomplished using ELISAs allows for smaller sample volumes and the detection of low abundance proteins. ELISAs generally provide an indirect measurement of kinase activity. However, variations in the technique described above use an immobilized capture antibody, substrate, and a phospho-substrate detection method for more direct measurements of kinase activity.

Box 2. R&D Systems Surveyor™ IC ELISA Kits provide all of the components necessary for running a successful ELISA, including cell lysis buffer, antibody-coated microplates, wash buffers, and a protein standard.
Phosphorylated ERK1/ERK2 in HeLa Cells. HeLa cells were treated with PMA in the presence or absence of MEK1/2 inhibitors U0126 or PD98059. Phospho-ERK1/ERK2 assessed in lysates generated from treated and untreated cells was quantified with the phospho-ERK1/ERK2 Surveyor IC Immunoassay kit (Catalog # SUV1018). The presence of similar levels of phosphorlyated and total ERK1/ERK2 was detected by Western blot using anti-phospho-ERK1/ERK2 affinity-purified polyclonal antibody (Catalog # AF1018) or anti-ERK1/ERK2 monoclonal antibody (Catalog # MAB1576).
The DuoSet IC ELISA Development Systems offer an economical alternative to complete kits by providing the essential components required for developing an assay. DuoSet IC Development kits include antibodies for capture and detection, protein standard or control for calibration, and Streptavidin-HRP.
Phosphorylated JNK in IL-1 beta-treated HepG2 Cells. Human hepatocellular carcinoma (HepG2) cells were treated with recombinant human IL-1 beta (Catalog # 201-LB) for the indicated times. Cell lysates were assayed for JNK phosphorylation using the Phospho-JNK DuoSet IC ELISA (Catalog # DYC1387; histogram). The results are consistent with the total amounts of phospho-JNK detected in the same lysates by Western blot using rabbit anti-human/mouse/rat phospho-JNK (T183/Y185) (Catalog # AF1205) or rabbit anti-human/mouse/rat JNK (Catalog # AF1387) polyclonal antibodies.

Cell-Based ELISA

Although in vitro biochemical kinase assays such as the typical sandwich ELISA are routinely used for hypothesis testing and drug screening, they cannot replicate the intracellular environment. Analyzing protein phosphorylation within intact cells may more accurately represent the status of specific signaling networks. Several immunoassays enabling the measurement of protein phosphorylation in the context of a whole cell have recently been developed. The cells are stimulated, fixed, and blocked in the same well. Phospho-specific antibodies are used to assess phosphorylation status using fluorometric (Box 3) or colorimetric detection systems. These assays bypass the need for the creation of cell lysates and are therefore more amenable to high throughput analyses.

Box 3. R&D Systems Cell-Based ELISAs are the first fluorometric assays to permit the simultaneous screening of both phosphorylated and total proteins in the same microplate well. Normalizing the fluorescence signal derived from the phospho-protein to that of the total protein makes it easy to account for potential well-to-well variabilities such as differences in cell number.

Measurement of ERK1/ERK2 (T202/Y204) Phosphorylation in A431 Cells. A431 human epidermoid carcinoma cells were treated with recombinant human (rh) EGF (Catalog # 236-EG) for the indicated times (A) or pretreated for one hour with the indicated concentrations of the MEK inhibitor U0126, and then incubated with or without rhEGF (B). After fixation of cells, phosphorylation of ERK1/ERK2 (T202/Y204) was determined and normalized to total ERK1/ERK2 in the same well using the Phospho-ERK1/ERK2 (T202/Y204) Cell-Based ELISA Kit (Catalog # KCB1018). Values represent mean ± range of duplicate determinations. Analysis of ERK1/ERK2 phosphorylation and total ERK1/ERK2 by Western blotting is also shown (inset A).

Intracellular Flow Cytometry and ICC/IHC

The traditional techniques of intracellular flow cytometry and immunocytochemistry/immunohistochemistry (ICC/IHC) are powerful tools for detecting phosphorylation events.^8,9 Flow cytometry uses a laser to excite the fluorochrome used for antibody detection. Filter sets and fluorochromes with non-overlapping spectra must be carefully chosen when assessing multiple proteins in the same cell. Flow cytometry is advantageous because it allows for rapid, quantitative, single cell analysis (Figure 2).¹⁰ Proteins can be detected in a specific cell type within a heterogeneous population via cell surface marker phenotyping without the need to physically separate the cells. In this way, a small, rare population of cells may be analyzed without concern for cell loss or altered protein expression that may occur during a cell-sorting process.

Figure 2. Detection of IL-4-induced STAT6 Phosphorylation by Intracellular Flow Cytometry. Human Daudi lymphoblastoid cells, (A) untreated or (B) activated with recombinant human IL-4 (Catalog # 204-IL) were fixed and permeabilized with methanol. Simultaneous detection (orange) of total (x axis) and phosphorylated (y axis) STAT6 was performed by co-staining cells with allophycocyanin-conjugated anti-STAT6 (Catalog # IC2167A) and phycoerythrin-conjugated anti- phospho-STAT6 (Catalog # IC3717P), respectively. The percent of cells positive for phospho-STAT6 is indicated in each treatment (upper right). Staining with isotype controls (Catalog # IC003A and # IC105P; maroon) highlights the specificity of the STAT6 antibodies.

ICC generally refers to protein detection by microscopy in cultured cells, while IHC refers to protein detection in intact tissue sections. Like flow cytometry, these techniques allow for the assessment of multiple proteins within a cell or tissue provided that adequate attention is given to avoid overlapping fluorescence spectra or color. Both fluorescent and colorimetric detection techniques are commonly used (Figure 3). In contrast to other formats for monitoring phosphorylation, ICC is usually the method of choice for determining intracellular localization. Both flow cytometry and ICC/IHC require high-affinity and high-specificity antibodies, blocking steps, controls, and antibody titration to eliminate ambiguous results resulting from non-specific binding.

Detection of phospho-proteins by flow cytometry and ICC require that the protein is stable and accessible to the antibody. Cells are usually stimulated and fixed with formaldehyde or paraformaldehyde to cross-link the phospho-proteins and stabilize them for analysis. The fixed cells must then be permeabilized to allow for entry of phospho-specific antibodies into the cells. Different permeabilization techniques are often useful for various subcellular locations. A mild detergent will allow for detection of cytoplasmic proteins, while alcohol may be required for antibody access to nuclear proteins. Alcohol permeabilization may also enhance phospho-protein detection using peptide specific antibodies due to the denaturing property of alcohol.¹⁰ For more detailed ICC/IHC protocols, please visit our website at www.RnDSystems.com/go/IHCProtocol.

Figure 3. Detection of Phosphorylated Proteins Using ICC/IHC. Human Daudi lymphoblastoid cells were treated with (A) recombinant human IL-4 (Catalog # 204-IL) or (B) left untreated. Phosphorylated STAT6 was detected using anti-human phospho-STAT6 polyclonal antibody (Catalog # AF3717), followed by staining with NorthernLights™ 557-conjugated goat anti-rabbit IgG (Catalog # NL004; red) and DAPI nuclear staining (blue).
	C: Phosphorylated ERK1/ERK2 was detected in a section of inflamed rat brain cortex using anti-human/mouse/rat phospho-ERK1/ERK2 polyclonal antibody (Catalog # AF1018). The tissue was stained with the anti-rabbit HRP-DAB Cell and Tissue Staining Kit (Catalog # CTS005; brown) and counterstained with haematoxylin (blue).

Mass Spectrometry

A comprehensive assessment of protein phosphorylation (phosphoproteomics) in complex biological samples, such as cell lysates, is important for understanding phosphorylation-based signaling networks. Large-scale phospho-protein analysis in complex protein mixtures involves identification of phospho-proteins and phosphopeptides and sequencing of the phosphorylated residues. Mass spectrometry (MS) techniques are useful tools for these tasks. Although MS can be used with excellent sensitivity and resolution to identify a single protein, there are several inherent difficulties for the analysis of phospho-proteins. First, signals from phosphopeptides are generally weaker, as they are negatively charged and poorly ionized by electrospray MS, which is performed in the positive mode.¹¹ Second, it can be difficult to observe the signals from low-abundance phospho-proteins of interest in the high-background of abundant non-phosphorylated proteins. To overcome these drawbacks, several enrichment strategies for phospho-protein analysis by MS have been developed including immobilized metal affinity chromatography (IMAC),¹² phosphospecific antibody enrichment (Box 4),¹³ chemical-modification-based methods such as beta-elimination of phospho-serine and -threonine,¹⁴ and replacement of the phosphate group with biotinylated moieties.¹⁵

Multi-Analyte Profiling

Box 5. R&D Systems Proteome Profiler™ Arrays allow for the measurement of multiple proteins in a single sample. These arrays require no specialized equipment and eliminate the need for multiple immunoprecipitation/Western blot experiments. Among the Proteome Profiler line are three kits designed to assess phosphorylated proteins: the human Phospho-RTK Array (Catalog # ARY001), the human Phospho-MAPK Array (Catalog # ARY002), and the human Phospho-Immunoreceptor Array (Catalog # ARY004). Array kits contain buffers, detection antibodies, and membranes spotted in duplicate with capture antibodies carefully selected for their specificity. For performing experiments, membranes are blocked and then incubated with lysate samples. Levels of phosphorylated protein are assessed using phospho-specific or phospho-tyrosine antibodies followed by chemiluminescent detection. Proteome Profiler Arrays have recently been used to study intracellular pathways involved in cytotoxicity,17 angiogenesis,18 and cancer.19-21

Effect of MEK1/2 Inhibitor U0126 on the ERK Pathway. Proteome Profiler Human Phospho-MAPK Array (Catalog # ARY002) was incubated with lysates from HeLa cells treated with PMA in the (A) presence or (B) absence of MEK1/2 inhibitor U0126. Graphical representation of the data is shown in (C).

Mass spectrometric techniques such as collision-induced dissociation (CID) and electron transfer dissociation (ETD) provide comprehensive parallel analysis of peptide sequences and post-translational modifications such as phosphorylation.¹⁶ These techniques are labor-intensive, and strategies for comprehensive phosphorylation analysis may not be needed if particular pathways are of primary interest. This has led to the development of several novel methods for measuring protein phosphorylation of multiple analytes simultaneously. In general, these involve the use of phospho-specific antibodies and include microplate-based, bead-based, and membrane-based detection formats. The obvious benefit of these assays is that throughput capability is greatly enhanced by bypassing the need for running multiple individual Western blots or traditional ELISA-based assays. These techniques are also known for providing more data while requiring very little sample volume. In trade, protein profiling assays are typically recognized as being less sensitive than their more conventional counterparts due to potential antibody cross-reactivity.

Conclusion

Assessing protein phosphorylation is often an essential component of the cell biologist's repertoire for understanding intracellular factors underlying cellular activities. Given the important role kinases play, it is critical for researchers to have quality tools for measuring protein phosphorylation and/or kinase activity. Each technique excels in different contexts, and care must be taken to choose the method that best fits the experimental design (Table 2). This review provides a brief glimpse of several of the most widely used methods for assessing protein phosphorylation. Because of a growing demand, methodologies continue to improve, bringing researchers closer to understanding these complex and important processes that ultimately control cellular function.

References

1. Sefton, B.M. & S. Shenolikar (1996) Analysis of Protein Phosphorylation. In Ausubel, F.M. et al. (eds): Current Protocols in Molecular Biology, New York: John Wiley & Sons, Inc. 18.1.1-18.1.5.
2. Ubersax, J.A. & J.E. Ferrell (2007) Nat. Rev. Mol. Cell Biol. 8:530.
3. Ni, Q. et al. (2006) Methods 40:280.
4. Johnson, S.A. & T. Hunter (2005) Nat. Methods 2:17.
5. de Graauw, M. et al. (2006) Electrophoresis 27:2676.
6. Ross, A.H. et al. (1981) Nature 294:654.
7. Czernik, A.J. et al. (1991) Methods Enzymol. 201:264.
8. Zell, T. et al. (2001) Proc. Natl. Acad. Sci. USA 98:10805.
9. Willinger, T. et al. (2005) J. Immunol. 175:5895.
10. Krutzik, P.O. et al. (2004) Clin. Immunol. 110:206.
11. Mann, M. et al. (2002) Trends Biotechnol. 20:261.
12. Brill, L. M. et al. (2004) Anal. Chem. 76:2763.
13. Steen, H. et al. (2002) J. Biol. Chem. 277:1031.
14. Zhou, H. et al. (2001) Nat. Biotechnol. 19:375.
15. Oda, Y. et al. (2001) Nat. Biotechnol. 19:379.
16. Molina, H. et al. (2007) Proc. Natl. Acad. Sci. USA 104:2199.
17. Zampieri, C.A. et al. (2007) J. Virol. 81:1230.
18. Cohen, E.E. (2006) Cancer Res. 66:6296.
19. Fujikawa, T. et al. (2007) J. Biol. Chem. 282:8741.
20. Menedez, J.A. et al. (2006) J. Clin. Oncol. 24:3735.
21. Engelman, J.A. et al. (2007) Science 316:1039.