Protein kinases transfer a phosphate group from ATP to the serine, threonine or tyrosine residues on the peptide substrate, directly affect the objectives’ activity and function. Radiological studies have shown that about 30% of the proteins in eukaryotic cells have the Phosphorylation phenomenon. This critical post-translational modification regulates a wide range of cellular activity, including cell cycle, differentiation, metabolism and neuronal communication. In addition, abnormal phosphorylation has relation to many diseases. There are many methods to assess phosphorylation, depending on several factors including the specific issues raised, as well as the availability of special equipment or reagents. This article briefly describes several common methods to detect protein phosphorylation, and presents the advantages and disadvantages of each method.
Kinase activity assay
Protein kinases are common component of many signal transduction networks. They affect on the downstream effectors which are responsible for many biological reactions. Therefore, assessing a particular kinase’s activity may provide valuable information for the parallel pathways. Kinase’s activity in a biological sample is typically determined in vitro by the incubation of kinase immunoprecipitate and the exogenous substrates, together with the presence of ATP. Then, adopt some of the reporting system, including chromogenic, radioactive or fluorescent detection, to assess the specific kinase phosphorylation of substrates. In addition, R & D Systems also provides a non-radioactive Universal Kinase Activity Kit, which can quantify activity of any ADP-producing kinases. Although we were able to obtain information about a particular kinase’s acts, but assessment of kinase’s activity in cell extract only reverses a tip of the iceberg of the whole cell’s signal pathway. We still know little about how the protein is modified, and in vitro activity analysis can not explain the potential role of endogenous phosphatase’s activity. Direct detection of phosphorylated proteins may provide a more detailed analysis about how cell response to external stimuli, because the identification of phosphorylated peptides provides information of protein’s expression and functional status.
Phospho-specific antibody development
A classical method for the direct determination of protein phosphorylation is: (1) incubation of the whole cell together with radiolabeled 32P-phosphate; (2) obtain the cell’s extracts, (3) separate the extracts by SDS-PAGE; (4) exposed on film. This method requires cumbersome, repetitive, several-hour incubation, and the use of radioisotopes. 2D gel electrophoresis is another traditional method. This technology based on the assumption that phosphorylation will change the protein’s mobility and isoelectric point.
Because these traditional methods are laborious, phospho-specific antibodies development is greatly welcomed by researchers. In 1981, the first recorded phosphorylated antibodies produced in rabbits, using phosphate binders of keyhole limpet hemocyanin (KLH) and benzoyl. This antibody can widely recognize the protein containing phosphorylated tyrosine. Ten years later, the use of synthetic phosphopeptides to immunize rabbits has improved the development of a number of phospho-specific antibodies. The phosphopeptides represent the amino acid sequence around the targeted protein’s phosphorylation sites. Then, load the immune serum into the peptide affinity column, forming a kind of highly specific immunological reagents. The appearance of phospho-specific antibodies improves traditional and become a basement for new immunoassay technology. The key for using phospho-specific antibodies is specificity of the antibodies and affinity between the antibodies and targeted phosphorylated proteins.
Western blot is the most commonly used methods to assess protein phosphorylation status. Most of the cell-biology laboratories have undertaken this method. Use the SDS-PAGE separation for the biological sample, and then transfer to a membrane (typically nylon or PVDF membrane). After that, identify the target protein by phospho-specific antibody. A typical Western blot test can avoid the dangerous-waste disposal requirements when using radioisotopes. Many phospho-specific antibodies are very sensitive and can easily detect the conventional phosphorylated protein samples (e.g. 10-30 μg cell extracts). Since the level of phosphorylated proteins may change with the processing errors or gel loading errors, researchers often use an antibody to detect the overall level of homologous proteins (regardless of phosphorylation status), in order to determine the relative proportion of phosphorylated components in the total components, as well as to serve as a loading internal control. CLIA and chromogenic methods are commonly used, and weight markers are used to provide information of the molecular weight of the protein molecules.
Enzyme-linked immuno sorbent assay (ELISA)
ELISA has become a powerful method for measuring protein phosphorylation. The method of ELISA’s quantitative ability is superior to Western blot, and plays a huge role in the study of the regulation of protein kinase’s activity and function. This microplate analysis generally use protein specific capture antibody, regardless of phosphorylation state. Then, let the target protein bound to the antibody-coated assay plates. The protein can be pure or a component in complex heterogeneous sample (e.g. cell lysate). Add specific antibodies containing specific phosphorylation site to analyze. These analyses are typically designed as chromogenic or fluorescent detection. Generated signal strength is proportional to the concentration of the phosphorylated proteins in initial samples. Compared with the more conventional immunoblotting methods, phospho-specific ELISA technique has several advantages. First, the use of calibrated standards makes the results be easily quantified. Secondly, in order to bring a high specificity, form a sandwich of two antibodies specific to the target protein. Third, ELISA has higher sensitivity and allows the use of smaller amounts of sample. It can also achieve the detection of low abundance proteins. Finally, the flux of microplate is much higher than conventional Western blotting. ELISA is usually use in indirect measurement of kinase’s activity. However, another type of ELISA technique using immobilized captured antibody, substrate and the phosphorylated substrate to directly detect the kinase’s activity.
Although extracorporeal biochemical kinase assays (e.g. a typical sandwich ELISA) are commonly used in the hypothesis testing and drug screening, they can not replicate the environment within the cell. Analysis of protein phosphorylation within the intact cells may be able to more accurately represent the status of the specific-signal-pathway network. Some immunoassays have recently been developed, which can measure protein phosphorylation in intact cells. Cells are stimulated, fixed and blocked at the same hole on the plate. Use fluorescent or chromogenic detection system with phospho-specific antibodies to assess the phosphorylation state. In addition, simultaneously detect the phosphorylated protein and total protein in the same hole of microplate. Thus, the signal from the target protein can be standardized by a second protein, correcting the differences between the holes to accurately assess the level of phosphorylated proteins and compare multiple samples. This method is similar to the traditional immunoblots using phospho-specific and total protein antibodies. These analyses avoid the requirements of preparing the cell lysates, and therefore are more suitable for high-throughput analysis.
Cellular flow cytometry and ICC / IHC
A traditional cellular flow cytometry and immunocytochemistry / immunohistochemistry (ICC / IHC) are powerful tools to detect phosphorylation. Flow cytometry (FCM) using laser to excite the fluorescent dyes for antibody detection. When evaluating a plurality of proteins in the same cell, optical filter combination and fluorochrome must be carefully selected to avoid the spectrum overlap. Flow cytometry have great advantages, because it achieves rapid and quantitative single cell analysis. By classifying cells’ surface marker, proteins of specific cell types can be detected in a heterogeneous group, without physically separating the cells. By this method, people can analyze rare cell groups, without the fear of losing cell protein or the change of protein expression in the process of cell sorting.
ICC generally refers to the use of the microscope to detect proteins in the cultured cells, while IHC refers to detect proteins in the intact tissue sections. Similar to flow cytometry, these technology assess a plurality of proteins within cells or tissues. Just remind that pay attention to avoiding overlaps of fluorescence spectra. Fluorescent and chromogenic detection techniques are often used. Different from other methods of detecting phosphorylation different, ICC is generally the preferred method for determining the intracellular localization. Flow cytometry and ICC / IHC all require high affinity, high specificity of the antibodies, blocking steps, antibody titration and control experiment to avoid ambiguous results due to nonspecific binding.
Using ICC and flow cytometry to detect phosphorylated protein requires the tested protein is stable and approachable for antibodies. Usually, after stimulation, fixation by formaldehyde or paraformaldehyde, and cross-bonding with phosphorylated proteins to improve the stability of cells. In this way, make convenience for analysis. After fixation process, cells must be permeabilized, so that the phospho-specific antibodies can enter cells. For different subcellular units, usually apply different permeabilization techniques. Mild detergent can help detection of cytoplasmic proteins, but for antibodies to approach nucleoprotein, ethanol will be required. Alcohol may also enhance the detection of phosphorylated proteins by phospho-specific antibodies, because alcohol has the denaturation.
A comprehensive assessment (phosphorylation proteomics) of protein phosphorylation in complex biological samples (such as cell lysate) is very important for understanding the phosphorylation-based signal network. Large-scale analyses in complex proteins mixture, including identification of phosphorylated proteins and phosphorylated peptides, as well as the measurement of amino acid residues’ sequence, are also play significant roles. Mass spectrometry (MS) technology is a useful tool to accomplish these tasks. Although MS has excellent sensitivity and resolution in identification of a particular protein, but there are still some inherent difficulties introducing MS into the protein phosphorylation analysis. Firstly, the signal of phosphopeptides generally is weak. Usually, they are negatively charged, but electrospray ionizsation mass spectrometry is working in positive electricity mode, so the phosphopeptides is not effectively ionized. Secondly, under the background of the great number of non-phosphorylated proteins, it is difficult to observe the low-abundance protein’s signal. To overcome these disadvantages, people have developed a number of enrichment strategies, including immobilized metal affinity chromatography (IMAC), phospho-specific antibodies enrichment, chemical modification (e.g. phosphorylation of serine and threonine β- elimination reaction), and using biotinylated groups to substitute phosphate groups.
Multiple map analysis
Mass spectrometry techniques, such as collision induced dissociation (CID) and electron transfer dissociation (ETD), provides a full parallel analysis of peptide sequence and post-translational modification (phosphorylation). This technology is quite laborious, and when a particular path is the main research objective, the full phosphorylation analysis strategy may not be needed. This result in a number of new methods to simultaneously determinate multiple protein phosphorylation analytes. In general, these methods come down to the usage of phospho-specific antibodies, including those based on microplates, magnetic beads and membranes. One of the best benefits of this analysis is greatly improving the throughput capacity, in order to avoid running multiple conventional ELISA or Western blot analysis. These techniques can also provide additional data, and require little sample volume. Accordingly, the protein multiple map analysis is generally considered less sensitive than conventional techniques, for the potential cross-reactivity of the antibodies.
Phosphorylated protein multiple assays
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Assessment of protein phosphorylation is an important part of cell biology, which can help people to understand the intracellular activity of cytokines. Taking the important role played by the kinase into account, researchers must have high-quality tools to determine the protein phosphorylation and the kinase’s activity. Each technology has advantages under different contexts. It is necessary to carefully select the most suitable experiment method design. This review briefly introduces some of the most commonly used methods to assess protein phosphorylation. Due to growing demand, the methods have also been improved, allowing researchers to better understand these complex and important process, and ultimately control cell’s function.