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 [1, 2]. The terms “top” and “bottom” refer to the complexity of the analyte, namely the more complex “protein” and less complex “peptide.” In top‐down protein analysis, the intact protein is directly analyzed by mass spectrometer. Mass information and fragment ions are generated from the intact protein ions and are then used for direct protein identification and characterization. In comparison, bottom‐up method starts with digesting the protein into peptides by either chemical or enzymatic digestion. The peptide product is then analyzed by a tandem mass spectrometer, and the peptide molecular weight and fragmentation informa­ tion is matched back to the original protein or protein mixture. When a mix­ ture of proteins is analyzed by a bottom‐up method, it is also called shotgun proteomics, owing to the similarity to shotgun genomic sequencing.