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Profiling of Protein N-Termini and Their Modifications in Complex Samples

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Protein Terminal Profiling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1574))

Abstract

Protein N termini are a unique window to the functional state of the proteome, revealing translation initiation sites, co-translation truncation and modification, posttranslational maturation, and further proteolytic processing into different proteoforms with distinct functions. As a direct readout of proteolytic activity, protein N termini further reveal proteolytic regulation of diverse biological processes and provide a route to determine specific substrates and hence the physiological functions for any protease of interest. Here, we describe our current protocol of the successful Terminal Amine Isotope Labeling of Substrates (TAILS) technique, which enriches protein N-terminal peptides from complex proteome samples by negative selection. Genome-encoded N termini, protease-generated neo-N termini, and endogenously modified N termini are all enriched simultaneously. Subsequent mass spectrometric analysis therefore profiles all protein N termini and their modifications present in a complex sample in a single experiment. We further provide a detailed protocol for the TAILS-compatible proteome preparation from plant material and discuss specific considerations for N terminome data analysis and annotation.

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Acknowledgments

The authors thank Prof. Chris Overall (University of British Columbia, Vancouver, Canada) and his team for establishing the original TAILS workflow and continued open exchange of information concerning improvements and adaptations. This work is supported by funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC starting grant “ProPlantStress”, grant agreement No 639905, to PFH). JNK is a recipient of a Career Investigator Scholar award from the Michael Smith Foundation for Health Research.

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Authors and Affiliations

  1. Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Wilhelm-Johnen-Str, 52425, Jülich, Germany

    Fatih Demir, Stefan Niedermaier & Pitter F. Huesgen

  2. Centre for Blood Research, Department of Pathology & Laboratory Medicine, University of British Columbia, 2350 Health Sciences Mall, Vancouver, Canada, V6T 1Z3

    Jayachandran N. Kizhakkedathu

  3. Department of Chemistry, University of British Columbia, 2350 Health Sciences Mall, Vancouver, Canada, V6T 1Z3

    Jayachandran N. Kizhakkedathu

Authors
  1. Fatih Demir
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  2. Stefan Niedermaier
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  3. Jayachandran N. Kizhakkedathu
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  4. Pitter F. Huesgen
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Corresponding author

Correspondence to Pitter F. Huesgen .

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Editors and Affiliations

  1. Institute of Molecular Medicine and Cell Research, University Freiburg, Freiburg, Germany

    Oliver Schilling

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Demir, F., Niedermaier, S., Kizhakkedathu, J.N., Huesgen, P.F. (2017). Profiling of Protein N-Termini and Their Modifications in Complex Samples. In: Schilling, O. (eds) Protein Terminal Profiling. Methods in Molecular Biology, vol 1574. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6850-3_4

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  • DOI: https://doi.org/10.1007/978-1-4939-6850-3_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6849-7

  • Online ISBN: 978-1-4939-6850-3

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