In in vitro phosphorylation mass spectrometry, the target protein or mixture of proteins is first phosphorylated under laboratory conditions. This is usually achieved through the use of kinases and ATP. The phosphorylated protein sample is then subjected to mass spectrometry. Mass spectrometers are able to accurately measure the mass of a protein or peptide and identify phosphorylation sites by detecting changes in quality due to phosphorylation. The detailed steps are as follows:
Figure 1Phosphorylation quantitative proteomics analysis workflow.
1.Prepare protein samples:
First, you need to prepare a sample of your protein of interest. This can be a purified protein or a complex cell extract. Ensure proper protein quality and concentration.
2.Phosphorylation reaction:
In in vitro phosphorylation experiments, protein samples are mixed with kinase-digested drugs to mimic intracellular phosphorylation reactions. Typically, phosphorylation reagents contain phosphorylated substrates (typically ATP), kinase enzymes, and reaction buffers.
3.Reaction stops:
After a period of reaction, the phosphorylation reaction is usually stopped using some method. Stopping methods typically involve adding a thermogel sample buffer or heating the sample to stop the reaction.
4.Sample separation:
The phosphorylated protein sample needs to be separated, usually by SDS-PAGE electrophoresis, to separate the proteins by molecular weight.
5.Dyeing and cutting:
The isolated proteins are stained on the gel and can then be extracted from the gel by cleavage. The protein band of interest is cut off for mass spectrometry analysis.
6.Mass Spectrometry:
The extracted protein bands can be analyzed by mass spectrometry, typically by liquid chromatography coupled with mass spectrometry (LC-MS) to determine the phosphorylation site. Mass spectrometry data will be analyzed to determine which lysine residues are phosphorylated.
7.Data Explained:
The obtained mass spectrometry data was analyzed to determine the phosphorylation site and phosphorylation level of the protein. This requires the use of relevant analytical software and databases to identify phosphorylation sites.
The key advantage of in vitro phosphorylation mass spectrometry is that it can provide detailed information about the phosphorylation status and phosphorylation sites of proteins. By comparing phosphorylated and non-phosphorylated samples, researchers can identify specific sites of phosphorylation events and how these modifications affect the function of proteins.