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SILProNAQ: A Convenient Approach for Proteome-Wide Analysis of Protein N-Termini and N-Terminal Acetylation Quantitation

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Protein Terminal Profiling

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

Abstract

Protein N-terminal modifications have recently been involved in overall proteostasis through their impact on cell fate and protein life time. This explains the development of new approaches to characterize more precisely the N-terminal end of mature proteins. Although few approaches are available to perform N-terminal enrichment based on positive or negative discriminations, these methods are usually restricted to the enrichment in N-terminal peptides and their characterization by mass spectrometry. Recent investigation highlights both (1) the knowledge of the N-terminal acetylation status of most cytosolic proteins and (2) post-translational addition of this modification on the N-terminus of nuclear coded chloroplast proteins imported in the plastid and after the cleavage of the transit peptide. The workflow involves stable isotope labeling to assess N-acetylation rates followed by Strong Cation eXchange (SCX ) fractionation of the samples to provide protein N-terminal enriched fractions. Combined with mass spectrometry analyses, the technology finally requires extensive data processing. This last step aims first at discriminating the most relevant mature N-termini from the characterized peptides, next at determining its experimental position and then at calculating the N-terminal acetylation yield. Stable-Isotope Protein N-terminal Acetylation Quantification (SILProNAQ) is a complete workflow combining wet-lab techniques together with dry-lab processing to determine the N-terminal acetylation yield of mature proteins for a clearly defined localization.

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Acknowledgments

This work was supported by French National Research Agency (grant no. ANR-13-BSV6-0004). This study has benefited from the facilities and expertise of the SICaPS platform of I2BC (Institute for Integrative Biology of the Cell). We thank C. Espagne, V. Jung, S. Kanor, D. Sumpton for technical improvements of the protocol, J.-P. Scarpelli and J. Dumestier for the development of the EnCOUNTer tool, and all colleagues of the team for fruitful discussions.

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Authors and Affiliations

  1. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris Saclay, Bâtiment 21, Avenue de la Terrassee, 91198, Gif-sur-Yvette cedex, France

    Willy V. Bienvenut, Carmela Giglione & Thierry Meinnel

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  1. Willy V. Bienvenut
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  2. Carmela Giglione
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  3. Thierry Meinnel
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Correspondence to Willy V. Bienvenut .

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

    Oliver Schilling

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Bienvenut, W.V., Giglione, C., Meinnel, T. (2017). SILProNAQ: A Convenient Approach for Proteome-Wide Analysis of Protein N-Termini and N-Terminal Acetylation Quantitation. 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_3

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  • DOI: https://doi.org/10.1007/978-1-4939-6850-3_3

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