Radiolabel studies suggest that approximately 30% of proteins in eukaryotic cells are subject to phosphorylation. A classical method of measuring protein phosphorylation involves 2D gel electrophoresis or incubation of cells with radiolabeled 32P-orthophosphate, the generation of cellular extracts, separation of proteins by SDS-PAGE, and then exposure to film. These methods are labor-intensive and requires radioisotopes. The following summary provides a brief description of several methodologies currently used to assess phosphorylation.
Kinase Activity Assays
Kinase activity is usually measured in vitro by incubating the immunoprecipitated kinase with a substrate in the presence of ATP. Measurement of the phosphorylated substrate can be assessed by reporter systems including colorimetric, radioactive, or fluorometric detection.
Phospho-Specific Antibody Development
The development of phosphorylation-dependent antibodies has been used by many researchers. LifeTein developed many phospho-specific antibodies for researchers. Phospho-specific peptides representing the amino acid sequence surrounding the phosphorylation site of the target protein were first synthesized and then conjugated to keyhole limpet hemocyanin (KLH) for immunization. The immune sera will then be applied to a peptide affinity column to generate a highly specific immunoreagent. The successful detection is dependent on the specificity and affinity of the antibody for the phospho-protein of interest.
Many phospho-specific antibodies are very sensitive and can readily detect the phosphorylated protein in a routine sample. Both chemiluminescent and colorimetric detection methods are common, and molecular weight markers are also generally used to provide information about protein mass.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISAs generally provide an indirect measurement of kinase activity and is more quantitative than Western blotting. The phospho-specific ELISA technique can easily quantify the results by utilizing a calibrated standard. Using of two antibodies specific for the target protein employed together in the sandwich format will render high specificity. In addition, the microplate-based format of ELISAs allows for high throughput and smaller sample volumes and the detection of low abundance proteins.
Analyzing protein phosphorylation within intact cells may more accurately represent the status of specific signaling networks. Usually phospho-specific antibodies are used to assess phosphorylation status using fluorometric or colorimetric detection systems.
Intracellular Flow Cytometry and ICC/IHC
Flow cytometry is advantageous because it allows for rapid, quantitative, single cell analysis.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.
Mass spectrometry (MS) techniques are useful tools for identification of phospho-proteins and phosphopeptides and sequencing of the phosphorylated residues. MS can be used with excellent sensitivity and resolution to identify a single protein or peptide. Although signals from phosphopeptides are generally weaker, new technologies have beed developed to enrich the MS signals. The enrichment strategies include immobilized metal affinity chromatography, phosphospecific antibody enrichment, chemical-modification-based methods and replacement of the phosphate group with biotinylated moieties.
Multi-Analyte profiling involves the use of phospho-specific antibodies and include microplate-based and membrane-based detection formats. These assays provide more data while requiring very little sample volume. However these assays are less sensitive than the conventional methods due to potential antibody cross-reactivity.