Protease Specificity Profiling Using Synthetic Combinatorial Peptide Libraries and Mass Spectrometry

The substrate specificity of proteases largely determines their function and biological role. Methods that can profile a protease's specificity with high detail are valuable tools for protease research. Synthetic combinatorial peptide libraries offer a large array of potential substrates that can be tested in a single experiment. Here, we describe the design and synthesis of a combinatorial peptide library using the one-bead-one-compound approach, followed by treatment with a protease, i.e., Pro-Pro endopeptidase (PPEP), and subsequent LC-MS/MS and data analysis.