Methods for Detecting Protein Phosphorylation (2)
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 or colorimetric detection systems. These assays bypass the need for the creation of cell lysates and are therefore more amenable to high throughput analyses.
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.
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 . 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.
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 (
Multi-Analyte
Profiling
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. 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.
Web Guider
Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics
