shotgun proteomic analysis refers to a use of bottom-up proteomics techniques to study the whole proteins in a complex mixture, such as serum, urine, and cell lysates, etc. It utilizes the technology of high performance liquid chromatography (HPLC) in combination with mass spectrometry (MS) technology. The most distinctive feature of shotgun proteomics is that it enables identification and comparative quantification of a wide range of proteins at the same time with minimal protein separation needed. Protein mixtures are first digested by protease, and the resulting peptides are separated in HPLC, followed by tandem MS/MS analysis to identify the sequence of each peptide. The identified peptide sequences are compared with database, in searching for the corresponding protein identity.