Edwin Krebs did more than elucidate the energy-producing Krebs’ cycle. In 1992, Krebs and colleague Edmond Fischer received the Nobel Prize for their work in discovering reversible protein phosphorylation. Little did they know in those early days that phosphorylation is also a major player in many cellular processes, including signal transduction, cell cycle regulation, cell growth, apoptosis, and differentiation. After decades of study, scientists now know that the presence of a phosphate molecule—usually attached to threonine, serine, and tyrosine residues—can spell the difference between health and disease.
Research has revealed how cells respond to insulin, and how cancer can develop when phosphorylation goes awry. Other discoveries are showing that phosphorylation could be involved in mood disorders, diabetes, and Alzheimer’s disease.
Despite numerous important discoveries, studying phosphorylated proteins has never been easy. Traditionally, researchers used radioactive inorganic phosphorous isotopes (32P or 33P) to tag cellular phosphorylated proteins. But this only works for cells in culture. Also, adding radioactive isotopes induced DNA fragmentation, elevated p53 tumor suppressor protein levels, altered cell morphology, and caused cell cycle arrest or apoptosis. And in today’s environmentally aware society, developing laboratory protocols that don’t use radioactivity is encouraged. Fortunately, researchers now have other options.
One popular method is the use of antibodies that specifically bind phosphorylated proteins. These antibodies can be used as probes to detect phosphorylated proteins on a Western blot. Though using anti-phosphate antibodies is relatively straightforward, these antibodies can be difficult to produce. One common problem is that many phosphorylated proteins dephosphorylate after being injected into the antibody-producing animal. Still, many good quality antibodies are available for purchase.
With antibodies and other methods, detecting phosphorylated proteins can be hampered by their usual low abundance, hidden in a sea of high abundance proteins. Researchers have come up with several ways to amplify the signal of phosphorylated proteins. With phosphates being rather labile, they can easily be replaced with biotin molecules via chemical methods. The resulting proteins are then put through a column of avidin, which has a high affinity for biotin. These once-phosphorylated proteins can then be eluted and studied and characterized by a mass spectrometer, or other technique.
One such technique is the use of stains, made of a small molecule fluorophore, that are specific for phosphorylated proteins. While enrichments can be used prior to staining, the stain already has a high level of sensitivity and specificity. This is important with the increasing use of the microarray format that requires detection at the level of nano- and picograms of protein.
In your study of protein function, there’s no doubt that you’ll someday be wondering if your pet protein is phosphorylated. Take a look below at the products that can make answering that question a snap.