Shotgun proteomics refers to the use of bottom-up proteomics techniques to combine high-performance liquid chromatography and mass spectrometry to identify proteins in complex mixtures. The name is derived from the shotgun sequence in DNA and named for the shotgun's rapidly expanding, quasi-random firing pattern . The most common method of shotgun proteomics begins by digesting the proteins in the mixture and separating the resulting peptides using liquid chromatography. Peptides are then identified using tandem mass spectrometry. Shotgun Proteomics enables global protein identification and systematic profiling of the dynamic proteome. It also avoids the modest separation efficiency and loss of mass spectral sensitivity associated with intact protein analysis. Cells containing the desired protein complement are grown. Proteins are then extracted from the mixture and digested with proteases to produce a peptide mixture. The peptide mixture is then directly loaded onto a microcapillary column to separate peptides according to their hydrophobicity and charge. As peptides elute from the column, they are ionized and separated by m/z in the first step of tandem mass spectrometry. The selected ions undergo collision-induced dissociation or other process to induce fragmentation. The charged fragments are separated in the second stage of tandem mass spectrometry.With the human genome sequenced, the next step is the verification and functional annotation of all predicted genes and their protein products. Shotgun proteomics can be used for functional classification or comparative analysis of these protein products. It can be used in projects ranging from large-scale whole proteome to focusing on a single protein family. It can be done in research labs or commercially. the shotgun concept has expanded to only refer to a gel-free methodology consisting of direct loading onto online strong cation exchange (SCX)/reversed-phase (RP) liquid chromatography (LC) combined with MS/MS. Not a thing. (2D-LC-MS/MS); the field has expanded to include alternative fractionation such as gel-based SDS-PAGE, isoelectric focusing, and other LC separations of proteins and/or peptides.