Protein analysis by shotgun/bottom-up proteomics

Bottom-up protein analysis refers to the characterization of proteins by analysis of peptides released from the protein through proteolysis. When bottom-up analysis is performed on a mixture of proteins it is called shotgun proteomics, which provides an indirect measurement of proteins through peptides derived from proteolytic digestion of intact proteins. In a typical shotgun proteomics experiment, the peptide mixture is fractionated and subjected to LC-MS/MS analysis. Peptide identification is achieved by comparing the tandem mass spectra derived from peptide fragmentation with theoretical tandem mass spectra generated from in silico digestion of a protein database. Because peptides can be either uniquely assigned to a single protein or shared by more than one protein, the identified proteins may be further scored and grouped based on their peptides. One or more protein or peptide fractionation techniques can be applied prior to MS analysis and database searching.