St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.
Mass spectrometry is one of the most sensitive methods in analytical chemistry, and its application in proteomics has been rapidly expanded after sequencing the human genome. Mass spectrometry is now the mainstream approach for identification and quantification of proteins and posttranslational modifications, either in small scale or in the entire proteome. Shotgun proteomics can analyze up to 10,000 proteins in a comprehensive study, with detection sensitivity in the picogram range. In this chapter, we describe major experimental steps in a shotgun proteomics platform, including sample preparation in the context of studying protein-protein interaction, mass spectrometric data acquisition, and database search to identify proteins and posttranslational modification analysis. Proteome quantification strategies and bioinformatics analysis are also illustrated. Finally, we discuss the capabilities, limitations, and potential improvements of current platforms.