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

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

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.

Key words

  • Protease specificity
  • Proteolysis
  • Proteomics
  • Proteome-derived peptide library
  • Proteomic identification of protease cleavage sites

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Correspondence to Pitter F. Huesgen .

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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

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2078-6

  • Online ISBN: 978-1-0716-2079-3

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