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.

Solubilize 4 milligrams of lyophilized ¹⁵N and ¹³C enriched ERK-phosphorylated-Tau in 400 microliters of NMR buffer. Add 5% deuterium oxide for field-blocking of the NMR spectrometer and 1 millimolar TMSP as an internal NMR signal reference. Also, add 10 microliters of a 40x stock solution of a complete protease inhibitor cocktail.

Transfer the sample in a 5-milliliter NMR tube using an electronic syringe with a long needle or using a Pasteur pipette. Close the NMR tube using the plunger. Remove any air bubbles trapped between the plunger and the liquid by moving the plunger.

Place the NMR tube in a spinner. Use the appropriate gauge to adjust its vertical position in the spinner, such that most of the sample solution will be inside the NMR coil. Next, start the airflow in the NMR instrument by clicking "Lift" in the magnet control system window. Carefully place the spinner with the tube in the airflow at the top of the magnet bore, then, stop the airflow, and let the tube descend into place inside the probe head within the magnet.

After allowing the sample to equilibrate to 25 degrees Celsius, type 'atmm' in the command line to perform semi-automatic tuning and matching of the probe head to optimize power transmission. Then, click "Lock" in the magnet control system window to engage the field frequency lock of the spectrometer.

Start the shimming procedure to optimize the homogeneity of the magnetic field at the position of the sample by first typing 'topshim gui' on the command line to open the shim window. Then, click "Start" in the shim window. Check the residual B zero standard deviation to verify that the shims are optimal.

Next, calibrate the P1 parameter, which is the length of a proton radiofrequency pulse in microseconds. This parameter is necessary to obtain a 90-degree rotation of proton spin magnetization. Aim for the 360-degree pulse using a one-dimensional spectrum of water protons.

Adjust the frequency offset by setting the 01 parameter to the proton water frequency in the one-dimensional spectrum. Finally, start the acquisition of a one-dimensional proton spectrum by typing 'zg' in the command line.