Methods of monitoring protein or peptide digestion by a proteolytic enzyme, comprising contacting a protein or peptide with a proteolytic enzyme in the presence of a fluorescent dye such as epicocconone or a related dye under conditions which allow digestion of the protein by the proteolytic enzyme and monitoring the fluorescence of the mixture over time. The protein may be a recombinant protein having a tag such as 6X HIS, 10X HIS, FLAG, CBP, HSV, S- tag or HA; or fusion proteins such as GST or MBP. Further, the protein or peptide may be present in a complex protein/peptide mixture, for example a biological sample such as serum or plasma or an entire proteome. The monitoring can be performed on the reaction mixture in real-time or via sampling. The temporal decrease in fluorescence signifies digestion of the protein or peptide by the proteolytic enzyme and the data can be used to extract kinetic rate constants for th digestion. The invention also relates to kits for carrying out the method.
|Publication status||Published - 10 May 2007|