Identification of Protease Specificity Using Biotin-Labeled Substrates

BACKGROUND: Proteolysis constitutes a major post-translational modification. For example, proteases regulate the activation or inactivation of various proteins, such as enzymes, growth factors, and peptide hormones. Proteases have substrate specificity, and protease expression regulates the specific and regional activation or inactivation of several functional proteins. METHODS: We demonstrate a novel method for determining protease specificity through the use of MALDI-TOF mass spectrometry with biotin-labeled substrates. RESULTS: This method was able to determine the specificity of TPCK-trypsin, V8 protease, elastase and cyanogen bromide cleavage, and the results were similar to previous reports. In addition, the method can be used to measure crude samples, such as tumor extracts. CONCLUSION: We demonstrated that this method could identify protease specificity after simple processing, even for crude samples.