Phosphorylation is a significant post-translational modification (PTM) affecting a protein's shape, behavior within the cell, and function. Kinase and phosphatase enzymes, respectively, remove or add phosphate groups, mostly to serine, threonine, and tyrosine residues. These amino acids all contain a nucleophilic group that reacts with adenosine triphosphate (ATP), replacing an oxygen on the terminal phosphorous and ejecting adenosine diphosphate (ADP).
While kinases and phosphatases function in all living cells they are particularly active in eukaryotes, in which phosphorylation is one of the half-dozen or so significant post-translational modifications. Approximately 30% of proteins in eukaryotic cells are subject to phosphorylation, which implies the existence of a huge, nearly untapped avenue for pharmacologic intervention in nearly all serious human diseases.
This idea has not been lost on pharmaceutical companies, which have produced about 30 approved kinase inhibitor drugs. Small molecule tyrosine kinase inhibitors include imatinib (for treating leukemia), which blocks phosphorylation that benefits tumors, and gefinitib (for breast, lung cancers), which blocks signals that promote cancer cell proliferation.
Determining the extent and locations of phosphorylation is critical to understanding biochemical pathways in cells, particularly how cellular activity is activated or suppressed. Through phosphorylation, cells regulate growth, apoptosis, cell cycle progression, and signal transduction. As such the ubiquitous, constant addition and removal of phosphate from protein serves as a specific on/off switch for individual cell operations, and for the cell as a whole.
Researchers use several techniques for detecting and quantifying protein phosphorylation, including kinase activity assays, phospho-specific antibodies, Western blot, enzyme-linked immunosorbent assays (ELISA), cell-based ELISA, intracellular flow cytometry, mass spectrometry, and multi-analyte profiling.
Western blotting
Quantifying phosphorylation with Western blot methods is well-established, with vendors offering reagents and kits for distinguishing phosphorylated vs. non-phosphorylated proteins. One such seller, Advansta, offers tips for detecting phosphorylated proteins by Western blot.
Yet Western blots, though much improved, are still 40-year-old technology.
“Traditional Western blotting includes many manual, time-consuming steps including running a gel to separate the protein of interest, blot transfer, blocking, antibody incubations, washing, and detection,” says Susan Harrison Uy, scientific content marketing manager at ProteinSimple.
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The company’s Wes® is a capillary-based system that automates Western blot-style assays. Sample and assay reagents load onto a specially designed plate, along with a capillary cartridge, and are inserted into the instrument, in which all assay steps from sample loading, protein separation, immunoprobing, detection, and analysis are fully automated. Total run time is about three hours.
Wes is referenced in more than 400 publications, including a recent study from the Mayo Clinic analyzing cell signaling in human adipose tissue. Here investigators compared Wes to traditional Western blotting, concluding that the two methods were comparable and that Wes “required less sample than the traditional Western blot and less technician/assay time, while achieving high sensitivity and good reproducibility.”
Results and capabilities are the bottom-line driver for adopting automated versions of formerly manual workflows. Uy explains how Wes achieves this: “Wes requires a lower sample volume than traditional Western blotting techniques and detects proteins at the low picogram level, with three-hour run times vs. several days. And intra-assay coefficients of variability are below fifteen percent.”
Antibody generation
The number and diversity of phosphorylation-detecting protocols is itself a ripe area for research and development. For example, Rockland Immunochemicals offers a diverse set of antibodies and reagents for detecting phosphorylated targets with great precision through Western blot, flow cytometry, immunohistochemistry, and immunofluorescence microscopy.
Generating antibodies for specific-site phosphorylation can be a challenge, says vice president of client solutions David Chimento. “Our antibodies are produced by selecting unique immunogenic peptides specific to the phosphorylation site. Particularly difficult are dually phosphorylated sequences, an example being Rockland’s SMAD3 phospho S423/phospho S425 antibodies.”
In the case of a polyclonal antibody, the generated antisera contains antibodies that recognize both the peptide sequence (nonspecific) and the desired phosphorylated sequence (phospho-site specific), which is the one Rockland purifies. “We produce the phosphorylation site-specific antibody using multiple purification steps, typically using a combination of protein A and affinity purification against the phosphorylated peptide sequence, and then cross-adsorbing the antibodies that bind the un-phosphorylated peptide away as waste product,” Chimento says.
The final purified antibody binds to the desired protein sequence only when it is phosphorylated.
The process for making the monoclonal antibody follows the polyclonal production method up to the step of generating antisera. “Once the antisera satisfies our criteria, we proceed to fusion and make antibody-producing hybridomas,” Chimento adds. The challenge then becomes screening for positive antibody-producing clones against the phosphorylated peptide, then counter-screening against the un-phosphorylated peptide to remove those clones that are not specific for phosphorylation.
As noted, many techniques can be used to detect phosphorylated proteins. The overarching challenge in producing antibodies for this purpose is screening them for specificity. Manufacturers often use cell lysates or cell-based assays in which cells must be treated to elicit the desired or target phosphorylation. For example cells might be treated with a DNA-damaging agent before testing for, say, phosphorylated serine. Cancer cell lines may also incorporate these phosphorylations without pretreatment.
Image: This Western blot figure shows the use of the Rockland anti-ATM pS1981 antibody (p/n 200-301-400) to probe for activation of the double-stranded break sensor protein ATM. In the experiment, HEK293 cells were either untreated or treated with a DNA damaging agent zeocin for up to 4 hours. Cell nuclear lysates taken at time 0, 0.5, 1, 2, and 4 hr. The nuclear lysates were probed for phosphorylation of ATM of residue Serine1981 using Rockland’s anti- ATM ps9181 antibody. Additionally, the blot was probed with anti-ELG, a loading control protein for nuclear lysate. The data show that ATM is phosphorylated or activated by 30 min of zeocin exposure, is maximally activated by about 1 hour, and maintains an activated state for the duration of exposure to DNA damage.
Tricky quantitation
“Western blot is an effective way to probe for phosphorylation of a specific protein when a good antibody is available,” Chimento explains. “The challenge is that often phosphorylation is a labile event and it can be quite difficult to generate a good sample in which the modified protein is preserved.”
For example the cytosolic protein involved in AKT signaling is maximal at five minutes after stimulation, then is rapidly stripped of phosphorylation, making it difficult for researchers to capture the event without a very good experimental design. Other phosphorylated proteins are quite stable.
“In either case the sample should be maintained in a buffer with a protease and phosphatase inhibitor cocktail,” Chimento advises. “Conversely, another control experiment might involve treating the phosphoprotein with a commercially available phosphatase, which strips away phosphorylation, and demonstrating antibody specificity on that species.
Other techniques like flow cytometry, histochemistry, and cytochemistry, require that the protein is stable and the site should be antibody-accessible.
“Quantitation is a tricky business and usually a relative phosphorylation can be reported,” Chimento says. “Some immunoassays and platform immunoassay systems use multiplex analysis of phospho/non-phospho targets allowing for some level of quantification.”