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Sensitive Plant N-Terminome Profiling with HUNTER

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Book cover Plant Proteases and Plant Cell Death

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

Abstract

Protein N-termini provide unique and distinguishing information on proteolytically processed or N-terminally modified proteoforms. Also splicing, use of alternative translation initiation sites, and a variety of co- and post-translational N-terminal modifications generate distinct proteoforms that are unambiguously identified by their N-termini. However, N-terminal peptides are only a small fraction among all peptides generated in a shotgun proteome digest, are often of low stoichiometric abundance, and therefore require enrichment. Various protocols for enrichment of N-terminal peptides have been established and successfully been used for protease substrate discovery and profiling of N-terminal modification, but often require large amounts of proteome. We have recently established the High-efficiency Undecanal-based N-Termini EnRichment (HUNTER) as a fast and sensitive method to enable enrichment of protein N-termini from limited sample sources with as little as a few microgram proteome. Here we present our current HUNTER protocol for sensitive plant N-terminome profiling, including sample preparation, enrichment of N-terminal peptides, and mass spectrometry data analysis.

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Acknowledgments

This work was in part supported by funding from the Deutsche Forschungsgemeinschaft DFG (project SFB-1403–414786233).

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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, Forschungszentrum Jülich, Jülich, Germany

    Fatih Demir, Andreas Perrar, Melissa Mantz & 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, Melissa Mantz & Pitter F. Huesgen

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

    Pitter F. Huesgen

Authors
  1. Fatih Demir
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  2. Andreas Perrar
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  3. Melissa Mantz
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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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Demir, F., Perrar, A., Mantz, M., Huesgen, P.F. (2022). Sensitive Plant N-Terminome Profiling with HUNTER . 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_12

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  • DOI: https://doi.org/10.1007/978-1-0716-2079-3_12

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  • 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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