Enzymatic digestion of proteins in gels for mass spectrometric identification and structural analysis

Enzymatic digestion of proteins is a key technique used in protein identification. By combining the digestion with mass spectrometric detection, proteins at very low femtomole levels, and in some cases subfemtomole levels, can be identified. Typically, one- or two-dimensional SDS-PAGE is used to isolate the proteins of interest, followed by staining with Coomassie blue, digestion-compatible silver stain, or Sypro Ruby for detection. Two-dimensional (2-D) fluorescence difference gel electrophoresis (DIGE), which uses Cy3 and Cy5 dyes for detection, allows comparison of two different sample states in order to locate proteins that are up- or down-regulated. In each case, an in-gel digestion, usually tryptic, is used with mass spectrometry to identify these proteins of interest. For large numbers of gel spots, robotic digestion can save time and money.