Quantitation by Parallel Reaction Monitoring (PRM)
The Thermo Scientific Orbitrap ID-X ( or Exploris 480) mass spectrometer can determine absolute quantities of selected proteins in complex mixtures, such as a whole cell extract.
The proteins are identified by one or more peptides, using their elution time, peptide mass, and mass of fragmentation products of these selected peptides in the protein of interest.
The amount of the selected peptides is determined by comparison with an isotopically labeled synthetic versions of the selected peptides. This is referred to as parallel reaction monitoring (PRM).
Three steps identify the peptides
To identify a specific protein, one or more peptides are chosen for monitoring, preferably in a preliminary experiment which identifies suitable product fragments of the selected peptides. Three steps identify the peptides of interest from a complex mixture of peptides:
1 – chromatographic elution time (usually from a reverse phase or similar column)
2 – mass/charge ratio (m/z) of the peptide (parent m/z)
3 – mass/charge ratio (m/z) of collision products of the peptide. In a PRM we obtain all fragment ions and can quantify using one or several.
An isotopically labeled, heavy, version of the peptide is added to the digested protein mixture. The heavy peptide elutes at the same time as the native, light, peptide of interest. The instrument is set to detect the peptide of interest at the light mass and heavy mass, and then detects the light (native) and heavy (labeled) masses (m/z) of the peptide collision products.
The ratio of signals of the peptide collision products is used to quantitate the peptide, and hence protein of interest. Up to 300 peptides can be monitored in one run.
Several companies can synthesize the labeled peptides.
The same principle can be applied to drugs and metabolites for their quantification.