Shotgun proteomics is a method of identifying proteins in complex mixtures using a combination of high performance liquid chromatography comibined with tandem mass spectrometry. Proteins in the mixture are digested with an enzyme, typically trypsin, and the resulting peptides are separated by nanoscale liquid chromatography and directed into a mass spectrometer. A quadrupole ion trap mass spectrometer is used to fragment and detect the peptides. Comparison of peptide fragmentation spectra with theoretical spectra generated in protein databases is used to identify the peptides and, subsequently, the proteins from which they were derived. This type of workflow is most appropriate for discovery projects and may require significant up-front sample fractionation to obtain comprehensive proteome coverage.
In general, most samples are prefractionated in some way (cytosolic, nuclear, membrane, organelle preps or ion exchange), quantified and then precipitated prior to in solution digestion with trypsin or another enzyme.
- Protocol for in solution digestion, click here.