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Profiling Sequence Specificity of Proteolytic Activities Using Proteome-Derived Peptide Libraries

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Plant Proteases and Plant Cell Death

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2447))

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Abstract

Substrate sequence specificity is a fundamental characteristic of proteolytic enzymes. Hundreds of proteases are encoded in plant genomes, but the vast majority of them have not been characterized and their distinct specificity remains largely unknown. Here we present our current protocol for profiling sequence specificity of plant proteases using Proteomic Identification of Cleavage Sites (PICS). This simple, cost-effective protocol is suited for detailed, time-resolved specificity profiling of purified or enriched proteases. The isolated active protease or fraction with enriched protease activity together with a suitable control are incubated with split aliquots of proteome-derived peptide libraries, followed by identification of specifically cleaved peptides using quantitative mass spectrometry. Detailed specificity profiles are obtained by alignment of many individual cleavage sites. The chapter covers preparation of complementary peptide libraries from heterologous sources, the cleavage assay itself, as well as mass spectrometry data analysis.

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Author information

Author notes

  1. Fatih Demir

    Present address: Department of Biomedicine, Aarhus University, Aarhus, Denmark

Authors and Affiliations

  1. Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Jülich, Germany

    Fatih Demir, Maithreyan Kuppusamy, Andreas Perrar & Pitter F. Huesgen

  2. Cologne Cluster of Excellence on Aging-related Disorders, CECAD, Medical Faculty and University Hospital, University of Cologne, Cologne, Germany

    Andreas Perrar & Pitter F. Huesgen

  3. Department for Chemistry, Institute of Biochemistry, University of Cologne, Cologne, Germany

    Pitter F. Huesgen

Authors
  1. Fatih Demir
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  2. Maithreyan Kuppusamy
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  3. Andreas Perrar
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  4. Pitter F. Huesgen
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Corresponding author

Correspondence to Pitter F. Huesgen .

Editor information

Editors and Affiliations

  1. Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia

    Marina Klemenčič

  2. VIB-UGent Cntr for Plant Systems Biology, Ghent University, Ghent, Belgium

    Simon Stael

  3. ZEA-3 Analytik, Forschungszentrum Juelich, Juelich, Germany

    Pitter F. Huesgen

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Demir, F., Kuppusamy, M., Perrar, A., Huesgen, P.F. (2022). Profiling Sequence Specificity of Proteolytic Activities Using Proteome-Derived Peptide Libraries. In: Klemenčič, M., Stael, S., Huesgen, P.F. (eds) Plant Proteases and Plant Cell Death. Methods in Molecular Biology, vol 2447. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2079-3_13

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  • DOI: https://doi.org/10.1007/978-1-0716-2079-3_13

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2078-6

  • Online ISBN: 978-1-0716-2079-3

  • eBook Packages: Springer Protocols

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