In cases where we are interested in a limited number of proteins within complex proteomes, it is possible to perform a targeted analysis of these proteins. We use the PRM (Parallel Reaction Monitoring) method based on high-resolution mass spectrometry, which reduces the risk of interference. During this analysis, the mass spectrometer specifically filters peptides corresponding to the proteins of interest and fragments them to obtain MS2-type spectra. The most intense fragments of each spectrum are extracted to reconstruct the elution peak of the corresponding peptide and obtain quantification by integrating the area under the peak.
About a hundred proteins can be monitored simultaneously in a single analysis.
This method allows for the validation of potential biomarkers identified during global proteomic analyses. It is also an alternative to Western blot when antibodies are not available or when a large number of samples need to be analyzed.
Signal normalization is done by adding an internal standard (cytochrome C digest) or isotopically labeled synthetic peptides.
The use of high-purity synthetic peptides can also provide an absolute quantification of the protein in the sample (µg/mL) and not just a relative quantification between several samples.