Phosphorylation Outside the Body for Mass Spectrometry

In in vitro phosphorylation proteomics analysis, the target protein or protein mixture is first phosphorylated under laboratory conditions. This is typically achieved using kinases and ATP. The phosphorylated protein samples are then subjected to mass spectrometry. The mass spectrometer can accurately measure the mass of proteins or peptides and identify phosphorylation sites by detecting mass changes caused by phosphorylation. The detailed steps are as follows:

 

Protein Sample Preparation

First, the target protein sample needs to be prepared. This could be a purified protein or a complex cell extract. Ensure the quality and concentration of the protein are appropriate.

 

Phosphorylation Reaction

In in vitro phosphorylation experiments, the protein sample is mixed with kinase digestive drugs to simulate the phosphorylation reaction inside the cell. Usually, the phosphorylation reagent includes a phosphorylation substrate (usually ATP), kinase enzyme, and reaction buffer.

 

Reaction Stop

After the reaction has been carried out for a period of time, a method is usually used to stop the phosphorylation reaction. This often involves adding a hot gel sample buffer or heating the sample to stop the reaction.

 

Sample Separation

The phosphorylated protein sample needs to be separated, usually through SDS-PAGE electrophoresis to separate the proteins by molecular weight.

 

Staining and Cutting

The separated proteins are stained on the gel, and then can be extracted by cutting. The target protein bands will be cut out for mass spectrometry analysis.

 

Mass Spectrometry

The extracted protein bands can undergo mass spectrometry analysis, usually through liquid chromatography-mass spectrometry (LC-MS/MS), to determine phosphorylation sites. Mass spectrometry data will be analyzed to determine which lysine residues have been phosphorylated.

 

Data Interpretation

The mass spectrometry data obtained is analyzed to determine the phosphorylation sites and level of the protein. This requires the use of relevant analysis software and databases to identify the phosphorylation sites.

 

The key advantage of in vitro phosphorylation mass spectrometry is that it can provide detailed information about the phosphorylation status and sites of a protein. By comparing phosphorylated and non-phosphorylated samples, researchers can identify specific sites of phosphorylation events and how these modifications affect protein function.