Quantitation of proteins and peptides by Selective Reaction Monitoring
The Thermo Scientific TSQ Quantum Access Max 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 selected peptides to identify 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 selective reaction monitoring (SRM) or Multiple Reaction Monitoring (MRM).
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:
- chromatographic elution time (usually from a reverse phase column)
- mass/charge ratio (m/z) of the peptide
- mass/charge ratio (m/z) of collision products of the peptide.
1. Elution time gives relatively little discrimination because a column may separate "only" 100 peptides.
2. A doubly charged form of the peptide is often measured. The mass/charge ratio, measured in the first stage of the mass spectrometer, narrows the number of possible proteins but is rarely adequate to uniquely identify a protein.
3. The fragmentation products of the peptide are called transitions. A preliminary experiment is desirable to chose the product ions for measurement, usually a y ion from the collision. The second stage of the mass spectrometer fragments by collisions with gas molecules the peptides selected in the first stage. The third stage measures the mass/charge ratio of selected fragments.
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.