Abstract
Global acetylation profiling (GAP) consists of heterologous expression of a given N-acetyltransferase (NAT) in Escherichia coli to assess its specificity. The remarkable sensitivity and robustness of the GAP pipeline relies on the very low frequency of known N-terminal acetylated proteins in E. coli, including their degree of N-terminal acetylation. Using the SILProNAQ mass spectrometry strategy on bacterial protein extracts, GAP permits easy acquisition of both qualitative and quantitative data to decipher the impact of any putative NAT of interest on the N-termini of newly acetylated proteins. This strategy allows rapid determination of the substrate specificity of any NAT.
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Acknowledgments
This work was supported by KatNat (ERA-NET, ANR-17-CAPS-0001-01) and CanMore (France-Germany PRCI, ANR-20 CE92-0040) grants funded by French National Research Agency (ANR). This work has benefited from the support of a French State grant (ANR-17-EUR-0007, EUR SPS-GSR) managed by the ANR under an Investments for the Future program (ANR-11-IDEX-0003-02), from the facilities and expertise of the I2BC proteomic platform (Proteomic-Gif, SICaPS), supported by IBiSA, Ile de France Region, Plan Cancer, CNRS and Paris-Saclay University, and from COST Action CA20113, supported by COST (European Cooperation in Science and Technology). We thank Lucile Jomat and Mahmoud Abdou (This team) for their contributions to the setting of robust plasmid and bacterial sample preparations, respectively.
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Meinnel, T., Boyer, JB., Giglione, C. (2023). The Global Acetylation Profiling Pipeline for Quick Assessment of Protein N-Acetyltransferase Specificity In Cellulo. In: Gevaert, K. (eds) Mass Spectrometry-Based Proteomics. Methods in Molecular Biology, vol 2718. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3457-8_8
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DOI: https://doi.org/10.1007/978-1-0716-3457-8_8
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