In-Depth Specificity Profiling of Endopeptidases Using Dedicated Mix-and-Split Synthetic Peptide Libraries and Mass Spectrometry

Analytical Chemistry, vol. 95, pp. 11621–11631

Abstract

Proteases comprise the class of enzymes that catalyzes the hydrolysis of peptide bonds, thereby playing a pivotal role in many aspects of life. The amino acids surrounding the scissile bond determine the susceptibility toward protease-mediated hydrolysis. A detailed understanding of the cleavage specificity of a protease can lead to the identification of its endogenous substrates, while it is also essential for the design of inhibitors. Although many methods for protease activity and specificity profiling exist, none of these combine the advantages of combinatorial synthetic libraries, i.e., high diversity, equimolar concentration, custom design regarding peptide length, and randomization, with the sensitivity and detection power of mass spectrometry. Here, we developed such a method and applied it to study a group of bacterial metalloproteases that have the unique specificity to cleave between two prolines, i.e., Pro-Pro endopeptidases (PPEPs). We not only confirmed the prime-side specificity of PPEP-1 and PPEP-2, but also revealed some new unexpected peptide substrates. Moreover, we have characterized a new PPEP (PPEP-3) that has a prime-side specificity that is very different from that of the other two PPEPs. Importantly, the approach that we present in this study is generic and can be extended to investigate the specificity of other proteases.

Authors 10

1. Bart Claushuis Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands

2. Robert A. Cordfunke Aachen Department of Immunology, Leiden University Medical Center

   Leiden University Medical Center

   Affiliation as printed

   Department of Immunology, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands

3. Arnoud H. de Ru Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands

4. Annemarie M. Otte Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands

5. Hans C. van Leeuwen

   Netherlands Organisation for Applied Scientific Research

   Affiliation as printed

   Department of CBRN Protection, Netherlands Organization for Applied Scientific Research TNO, Rijswijk, 2280 AA, The Netherlands

6. Oleg I. Klychnikov

   Lomonosov Moscow State University

   Affiliation as printed

   Department of Biochemistry, Moscow State University, Moscow 119991, Russian Federation

7. Peter A. van Veelen Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands

8. Jeroen Corver Aachen Department of Medical Microbiology

   Leiden University Medical Center

   Affiliation as printed

   Department of Medical Microbiology, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands

9. Jan Wouter Drijfhout Aachen Department of Immunology, Leiden University Medical Center

   Leiden University Medical Center

   Affiliation as printed

   Department of Immunology, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands

10. Paul J. Hensbergen corresponding Aachen Center for Proteomics and Metabolomics

    Leiden University Medical Center

    Affiliation as printed

    Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands