PMF (peptide mass fingerprint) method
Proteins are identified by matching the profiles of peptides obtained by protein digestion to those registered in a database.
This method is recommended for high-purity proteins.
When a high-purity protein is treated with a digestive enzyme such as trypsin, the protein is digested according to the specificity of the enzyme and is degraded into peptides whose types depend on the amino acid sequence of the protein. The peptide products are characteristic of the protein. Thus, a protein can be identified by measurement of the masses of multiple peptides obtained after digestion.
The measurement and analysis procedure is explained below using the example of a protein separated by two-dimensional electrophoresis.
- Cut out the protein spot to be analyzed from the two-dimensional electrophoresis gel.
- Destain the protein according to the staining method.
- Reduce and alkylate the protein (to inhibit disulfide bond formation).
- Digest the protein in the gel with trypsin and extract the digested peptides.
- Desalt the peptides (salt contamination in the sample inhibits ionization in mass spectrometry).
- Perform mass spectrometry to obtain the actual measured values of mass data sets of the peptides.
- Search a protein database for theoretical values of MS/MS fragments of peptides obtained by trypsin digestion of proteins. Compare the theoretical values to the actual measured values, and select those that have a high relevance ratio.
Principle of the PMF method