How is Phosphorylation Level Determined?

The phosphorylation level of organic molecules or substances is determined by two methods: western blotting and mass spectrometry. Phosphorylation most often refers to the attachment of a phosphate to protein. When phosphates attach to protiens, the natural processes and functions of protein enzymes within the human body is disturbed. Determining the phosphorylation level is crucial to treating a wide range of diseases, from cancer to diabetes.

Western blotting is a popular method used to determine the phosphorylation level of a particular set of proteins. Antibodies such as anti-phospho-tyrosine monoclonal antibodies are typically used because they easily bind to a protein's tyrosine residues, which are affected by phosphorylation. In western blotting, the proteins are first separated by size using an electrical charge method known as gel electrophoresis.

The proteins are then transferred to a membrane that has been covered with special antibodies that bind to phosphates on proteins. Once the proteins are transferred, the antibodies will stick to any proteins with an attached phosphate. The phosphorylation level can then be determined by an x-ray of the membrane. Any affected proteins will give off a flash of light, making it easier for the researcher to measure phosphorylation levels.

Mass spectrometry is another way to measure the phosphorylation level of a substance. This process converts the protein molecules into ions, which makes them easier to track and measure. The sample is ionized by a high-powered ion source which inundates the sample with electrons. This action turns the sample into cations, positively charged ions.

The mass analyzer of the mass spectrometer separates the ions by their mass and intensity of charge. This allows the researcher to discover the mass-to-charge ratio, which is critical in discovering the phosphorylation level of the sample. Finally, the detector measures the ions in order so that the data can be displayed in chart form. The chart shows the mass-to-charge ratio of the ions on the x-axis, and the relative intensity of the ions on the y-axis.

Phosphorylation levels can also be measured during substrate phosphorylation. Substrate phosphorylation is a process that creates adenosine triphosphate (ATP), a molecule critical to energy transfer among cells, during glycolysis. This process occurs in both animal cells and plant cells.

In plant cells, ATP molecules are created by using energy from the sun. This energy spurs phosphorylation in the plant's chloroplasts, the site of plant photosynthesis. The level of phosphorylation correlates to the amount of phosphate needed to create additional ATP molecules.