Protein Analysis by Shotgun Proteomics

In mass-spectrometry-based protein analysis, there are two major strategies, the top-down method and the bottom-up method. When a mixture of proteins is analyzed by a bottom-up method, it is also called shotgun proteomics, owing to the similarity to shotgun genomic sequencing. This chapter discusses typical procedures of shotgun proteomics experiment and some advances regarding existing challenges. In a typical shotgun proteomics experiment performed on a modern instrument, one should expect to identify anywhere from 1000 to 10000 proteins from a mammalian cell lysate. A typical shotgun proteomics experiment consists of three main steps: sample preparation, mass spectrometry data acquisition, and data processing. Identification of peptides within the sample is accessed with the help of data-dependent acquisition on the basis of initially unbiased sampling. The most commonly used technique for peptide separation is nanoelectrospray along with reversed-phase nanoflow liquid chromatography.