Phosphoproteins: The Verbs of the Proteomic Language

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Phosphoproteins: The Verbs of the Proteomic Language

To fulfill the promise of the genome project, the proteomics project is needed
by Andrew J. Czernik, Michael D. Browning and John W. Haycock

click the image to enlarge
Figure 1. Design of phosphopeptide immunogen for the Ser-40 site on rat TH. The primary sequence of residues 27-53 of TH is shown in the middle of the figure. The 10-mer phosphopeptide immunogen is shown at the bottom of the figure.

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Figure 2. Immunization with phosphopeptide conjugates can lead to the presence of several antibody types in the antiserum. One can obtain the phosphospecific antibody that is desired (blue). However, it is also possible that one will obtain antibodies that are specific for the dephospho peptide (green) as well as antibodies that are pan-specific (red) and do not react with the protein in a phospho-specific manner.

The genome project offered the promise that new drugs could be discovered in a rational, direct and expedited fashion - rather than the more torturous approach used in previous decades. But the ink wasn't dry on the genome project before it was realized that in order to fulfill that promise, the proteomics project was needed. This is because protein function is what generally goes awry in disease and because proteins are the most common targets of drugs to treat those diseases. However the proteomics project is 10 to 30 times larger than the genome project. Thus the proteomics project represents an enormous challenge for bioscience.

To make this project more manageable, PhosphoSolutions is focusing on a specific subset of proteins known as phosphoproteins. While phosphoproteins represent only 10-20% of the total proteome, they are the most dynamic proteins in the body. They regulate such diverse processes as cell division in cancer and neuronal communication in memory and Alzheimer's disease. The significance of this area of study is reinforced by the fact that Nobel Prizes in two of the last three years were awarded to scientists for their work on phosphoproteins. Because protein phosphorylation regulates virtually every important cellular function, we consider phosphoproteins to be the verbs of the proteomics language.

This review will emphasize the importance of phosphoproteins as a focal point for proteomics research and describe the development of antibodies that bind to target proteins only when the protein is in the phosphorylated state. Thus phospho-specific antibodies can be used to assess the activity of a protein, not simply its level of expression.

History of phosphoprotein detection
Protein phosphorylation is the major cellular mechanism used to regulate protein function. As a class, phosphoproteins are substrates for an enzymatic, reversible post-translational modification known as phosphorylation, in which a phosphoryl group is covalently attached to or removed from the hydroxyl moiety of serine, threonine, or tyrosine residues. Phosphorylation regulates protein function by affecting conformation. This in turn regulates such processes as enzyme activity, protein-protein interactions, subcellular distribution, and stability and degradation. The stoichiometry of phosphorylation of a given site is controlled by the relative activities of a cell's repertoire of protein kinases and phosphatases. Thus phosphorylation can often generate extremely rapid and reversible changes in the activity of target proteins. The ability to assay the state of phosphorylation of specific proteins is of great utility in the quest to establish the function of a given protein. Such assays are also critical for the identification of drugs that can influence the phosphorylation, and hence the function of specific proteins.

In early studies, methods commonly used to measure protein phosphorylation and dephosphorylation in cell preparations employed prelabeling with 32Pi, in vitro phosphorylation with [g-32P] ATP, or "back" phosphorylation.(1,2) These methods continue to be very effective and have advantages for many test systems, but they do have several practical and theoretical limitations. Based in large part on the successful use of short synthetic peptides to produce epitope-targeted antibodies,(3,4) an immunochemical approach became an attractive alternative for detecting changes in the state of phosphorylation of specific proteins at a specific site. The use of phosphorylation state-specific antibodies takes advantage of the sensitivity and selectivity afforded by immunochemical methodology to greatly increase not only the throughput but also the quantitative accuracy of phosphoprotein assays.

Development of phospho-specific antibody-based assays
The first report of the production of phosphorylation-dependent antibodies appeared in 1981, when polyclonal antibodies that could detect phosphotyrosine-containing proteins were produced by immunization of rabbits with benzyl phosphonate conjugated to keyhole limpet hemocyanin (KLH).(5) These antibodies became key reagents in oncogenic virus and cancer research, but they were not selective for an individual protein or site of phosphorylation. Rather, they detected phosphotyrosyl moieties on many different proteins. Shortly thereafter, Nairn and colleagues reported the production of serum antibodies that distinguished between the phospho- and dephospho-forms of G-substrate, a protein localized to cerebellar Purkinje cells and phosphorylated by cGMP-dependent protein kinase.(6) A synthetic heptapeptide, Arg-Lys-Asp-Thr-Pro-Ala-Leu, corresponding to a repeated sequence surrounding two phosphorylated threonyl residues in the intact protein, served as antigen. Rabbit antisera against a peptide-KLH conjugate were specific for the dephospho-form of G-substrate. Phospho-specific antibodies were prepared by immunization of rabbits with the purified phosphoprotein, phosphorylated in vitro to a stoichiometry of 2 mol/mol with cGMP-dependent protein kinase. Despite this initial success, many other attempts to produce phospho-specific polyclonal antisera by immunization with the phospho-form of holoproteins were not very successful, probably because of two significant factors. First, many phosphorylated proteins are believed to undergo rapid dephosphorylation during immunization, regardless of the route of injection, leading to the loss of the desired phospho-epitope. Second, holoproteins generally contain multiple immunogenic epitopes; this decreases the probability that clonal dominance for a phospho-specific epitope will be obtained.

Taking a more direct approach utilizing phosphorylated and unphosphorylated forms of synthetic phosphopeptides, we developed a general protocol for the production of phosphorylation state-specific antibodies for substrates with established site(s) of phosphorylation.(7) In the early stages of development of this methodology, phosphopeptides were routinely prepared by enzymatic phosphorylation. At the same time, advances were being made in the chemical synthesis of phosphopeptides(8-10) and such phosphopeptides were being used to produce phospho-specific antibodies.(11) We also contributed to the refinement of methods for post-synthesis global phosphorylation to produce chemically phosphorylated peptides.(12,13) These enzymatic and chemical approaches remain perfectly valid today. However, the use of commercially available, high quality, affordable O-benzyl-protected Fmoc derivatives of phosphoamino acids has become the state-of-the-art in the preparation of synthetic phosphopeptides.(13) The production and use of phospho-specific antibodies has become an area of intense interest. Therefore, we describe below some of the important features involved in successfully producing phospho-specific antibodies.

Custom-made phospho-specific antibodies
The 30,000+ human genes predict that there will be thousands of physiological relevant phosphorylation sites. Technical advances in protein chemistry such as automated gas-phase N-terminal sequenators(14) and, more recently, the explosion in the use of mass spectrometry to assist in proteomics research have facilitated the ability to obtain direct proof that a potential site is phosphorylated in vivo.(15-17) Once the phosphorylation site of interest has been conclusively established, the development of custom-made, phospho-specific antibodies can proceed.

Design of the phosphopeptide antigen
The first step in developing custom-made, phospho-specific antibodies is the selection and design of the immunizing peptide. This is a crucial step because the specificity and utility of these antibodies depend critically on the design of the immunizing peptide. At PhosphoSolutions, Drs. John W. Haycock and Andrew J. Czernik - who together have more than three decades of experience in designing and producing phospho-specific antibodies - are responsible for the design of immunizing peptides.

As an example of this process, we have chosen to use the protein kinase A-dependent site of phosphorylation of the enzyme tyrosine hydroxylase (TH). TH catalyzes the rate-limiting step in the synthesis of the catecholamine neurotransmitters (e.g. dopamine). Importantly, phosphorylation at Ser-40 on TH increases the catalytic activity of the enzyme.(18) The sequence surrounding the phosphoserine is shown in the middle of the Figure 1, and the phosphopeptide designed for use in the production of the phospho-specific antibody is shown at the bottom of the figure. The rationale for the design of this peptide is as follows.

Truncated N-terminal sequence: The peptide has a truncated N-terminal sequence. This was done to eliminate reactivity with homologous proteins that were identified in a Blast homology search.

Use of a short sequence: Another feature of the peptide design was the use of a very short sequence surrounding Ser-40. This maximizes the probability that the phosphoseryl residue will be included as part of the major epitope to be recognized by the antibodies.

Amidation of C-terminus: The C-terminus was amidated to better emulate in the phosphopeptide the net charge in that region of the native TH protein. Such modifications can improve immunoreactivity in western blots and in immunohistochemical applications.

Addition of N-terminal cysteinyl residue: An N - terminal cysteinyl residue was added to permit easy and efficient conjugation to the carrier protein using sulfo-MBS and coupling to the commercially available affinity resin (Sulfo-Link resin, Pierce.) Addition of a cysteinyl residue at the N-terminal (or C-terminal) also assures that conjugation will occur at one end of the phosphopeptide, thus reducing the possibility that steric hindrance would interfere with access to the phosphorylated residue as part of the desired epitope.

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Figure 3. Sequential affinity chromatography is used to isolate phospho-specific antibodies. (See text for description.)

Conjugation and coupling of the phosphopeptide
The next stage in phospho-specific antibody production is peptide conjugation. This is a step that often receives little attention. However, it is a critical step in the production of phospho-specific antibodies. At PhosphoSolutions, we go to great lengths to ensure that both the conjugation of the phosphopeptide to carrier protein and the coupling of the peptides to affinity-purification resin are optimized. We carefully monitor the efficiencies of conjugation and coupling so that we can accurately control the immunization of rabbits and also obtain an optimal ratio of peptide to resin for the affinity-purification column. For example, high-affinity antibodies may not be effectively eluted if the ratio of phosphopeptide to resin is too high.

Phospho-antibody purification
Three antibody possibilities: Multiple steps of affinity chromatography can be required in order to isolate antibodies that are phospho-specific. This is due to the fact that a typical immunization with a phosphopeptide can generate three different types of antibodies, as depicted in Figure 2. The first is the phospho-specific antibody that is desired (in blue). However, antibodies that are specific for the dephospho form of the peptide (in green) can also be generated if phosphatases in the rabbit dephosphorylate the phosphopeptide conjugate that is injected. Lastly pan-specific antibodies (in red) can be generated against sequences/conformations of the phosphopeptide that do not involve the phosphoryl group in the epitope. These antibodies react with the protein irrespective of its phosphorylation state. The preferred way to isolate the desired phospho-specific antibody is through sequential phospho- and dephospho-affinity chromatography.

Isolation of the phospho-specific antibody: The first column we use is the phosphopeptide affinity column (see Figure 3a). An IgG fraction isolated from the antiserum is prepared using protein A affinity chromatography and then applied to our phosphopeptide affinity column. Two of the three types of antibodies that we described in Figure 2 will bind to this column. First, the phospho-specific antibody (in blue) will bind because the phosphopeptide is present on this column. The pan-specific antibody (in red) will also bind because the sequence/conformation of the phosphopeptide that it recognizes is also present on this column. The one antibody that will not bind to this column is the antibody (in green) that is specific for the dephospho form of the peptide, as this form of the peptide is not present on the column. These latter antibodies elute from the column in the flow-through, together with the entire complement of additional IgGs that were isolated from the rabbit antiserum. These "flow-through" antibodies are saved, and the phospho- and pan-specific antibodies are then eluted from the phosphopeptide affinity column. It is critical that elution buffers be optimized to induce release of the very high affinity antibodies from the column without irreversibly denaturing the antibodies. It is our experience that commonly used elution buffers such as 0.2M glycine (pH 3.0) often fail to meet these criteria.

The eluted pool of phospho- and pan-specific antibodies is then applied to the dephosphopeptide affinity column. Only the pan-specific antibodies bind to this column because the pan-specific peptide sequence epitopes but not the phosphopeptide epitope are present. The phospho-specific antibodies do not bind to the column and will be present in the flow-through (see Figure 3b). While the flow-through contains our desired phospho-specific antibodies and is saved, the pan-specific antibodies can be eluted and saved if they are present.

Characterization of the phospho-specific antibodies
The final stage of custom phospho-specific antibody production is the characterization of the antibodies. We use differential dot blots employing the phospho- and dephosphopeptides for our initial screens of the antisera. However, western blots are essential to truly establish the specificity of the antibodies for the holoprotein of interest.

Specificity of the antibodies: The first western blot analysis simply demonstrates that there are no cross-reacting bands labeled by the antibody in the sample of interest, such as a homogenate or cell lysate. Thus, as seen in a western blot of increasing concentrations of caudate nucleus homogenate using the phospho-Ser40 TH antibody, only a single band is labeled at the appropriate molecular weight in SDS-PAGE (Figure 4). The second level of specificity uses western blots of phospho- and dephospho-TH to demonstrate the selectivity of the phospho-specific antibody. As shown in Figure 5, the pan-specific antibody for TH recognized both the dephospho-TH and the phospho-TH. Most importantly, the phospho-specific antibody recognized only phospho-TH whereas the dephospho-specific antibody reacted selectively with the dephospho-TH.

Immunohistochemistry: Now we turn to immunohistochemistry, where the phospho-specific antibody enables visualization of the phosphorylation-dependent activation of a protein in situ. As illustrated in the left panel of Figure 6, the phospho-specific TH antibody shows virtually no labeling in this photomicrograph of a dark-adapted retina. In contrast, the phospho-specific TH antibody exhibits dramatic labeling of amacrine cells as is shown in the right panel of the figure.

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Figure 4. Western blot demonstrating the specificity of the phospho-Ser40-TH antibody. The antibody recognizes only a single band in a lysate from rat caudate. The single band migrates at the MW (60kD) appropriate for TH.

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Figure 5. Immunolabeling of phosphorylated and dephospho-TH. Identical amounts of phospho TH (Phos TH) and dephospho TH (Dephos TH) were run on western blots and then probed with either the pan-specific TH antibody (Anti-Pan), the phospho-ser40-TH specific antibody (Anti-phospho) or the dephospho-ser40-TH specific antibody (Anti-Dephospho).

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Figure 6. Immunohistochemistry of rat retina with the phospho-ser40-TH antibody. The photomicrograph at the left shows no labeling of the dark-adapted retina. However, as shown in the photomicrograph at the right, after brief light exposure of the retina, two amacrine cells exhibit strong immunolabeling with the Phospho-Ser40 TH antibody.

Future directions
As this summary indicates, we devote a great deal of care to the production of phospho-specific antibodies for an individual research scientist's protein of interest. However, there are also a number of limitations on the current use of phospho-specific antibodies. Principal among these is the fact that quantification of phosphorylation state using such antibodies typically can be performed on only a limited number of samples and a limited number of conditions. Also, a detailed analysis of multiple targets, such as clusters of related proteins in a given signal transduction cascade, is simply not feasible with current quantitative technology that involves the use of phospho-specific antibodies in western blots. Thus, the high-throughput demands associated with proteomics research must be addressed with new technological advances. To address this need we are proposing to develop protein microarray chips using phospho-antibodies as capture or detection reagents that should permit the simultaneous measurement of the phosphorylation state of dozens of distinct phosphoprotein targets in a single sample.

One might ask, why would one want such a complex data set. The answer to this question lies in the consensus that is emerging regarding the complexities of the cross talk among protein phosphorylation cascades that underlie normal physiology and disease states. It is becoming increasingly clear that protein phosphorylation can no longer be conceptualized in terms of single steps in a linear intracellular second messenger pathway activating a single protein kinase leading to modification of one specific protein. Rather there is overwhelming evidence that intracellular signaling involves multiple messengers that activate complex arrays of pathways, which intersect each other at many sites in a web-like fashion. Dissection of these phosphorylation networks is simply not possible with currently available technologies. To address this issue we are proposing to develop a phosphoprotein array platform that can permit us to address critical questions about the role of phosphoproteins within the grammar of disease.

About the authors
Dr. Andrew J. Czernik (ajc@phosphosolutions.com) is a Co-Founder and CSO of PhosphoSolutions. He received his Ph.D. in Biochemistry from New York Medical College and prior to joining PhosphoSolutions, Dr. Czernik was Senior Research Associate, Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY. Dr. Michael D. Browning is a Co-Founder and President/ CEO of PhosphoSolutions. He received his Ph.D. in Biology from the University of California. Dr. Browning is Professor of Pharmacology and Neuroscience at the University of Colorado Health Sciences Center in Denver, CO. Dr. John W. Haycock is a Co-Founder of PhosphoSolutions. He received his Ph.D. in Biology from the University of California at Irvine. Dr. Haycock is Professor of Biochemistry and Molecular Biology at Louisiana State University Health Sciences Center in New Orleans, LA.

More information on phosphoproteins and their applications is available from: PhosphoSolutions, Inc., Aurora, CO. 888-442-7100; phosphosolutions.com

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