Nuclear Magnetic Resonance Spectroscopy to Identify Multiple Phosphorylations in Proteins

Multiple phosphorylations in a protein involve adding phosphate groups to protein at various positions, specifically at serine and threonine residues.

To identify multiple phosphorylations in a protein, begin with a tube containing phosphorylated proteins labeled with nitrogen-15, which helps to differentiate amide hydrogens from other hydrogens in the protein.

Add an internal NMR reference compound for accurate measurement of the relative resonance frequencies. Transfer the mixture to a nuclear magnetic resonance, or NMR, tube. Insert the tube into a spinning aperture, and place it in an NMR spectrometer.

The presence of phosphates exhibits electron-withdrawing effects in the phosphorylated serine and threonine residues. As a result, the amide hydrogens of phosphorylated residues appear to be deshielded with lesser electron densities compared to the other amide hydrogens.

Amide hydrogens with varying electronic environments experience differential magnetic fields around nuclei, attaining low-energy states.

Apply a brief radiofrequency pulse to excite the hydrogen nuclei to a higher energy state at a specific resonance frequency based on their electronic environment.

During relaxation, the nuclei return to a low-energy state by releasing the absorbed energy, which is detected to produce an NMR signal.

Plot a two-dimensional NMR spectrum representing the individual amide hydrogens.

Compare the spectrum of the phosphorylated protein with that of the non-phosphorylated protein. The appearance of additional resonances in the phosphorylated protein’s NMR spectrum confirms multiple phosphorylations.