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An experimental strategy for the identification of AMPylation targets from complex protein samples

Pieles, Kathrin and Glatter, Timo and Harms, Alexander and Schmidt, Alexander and Dehio, Christoph. (2014) An experimental strategy for the identification of AMPylation targets from complex protein samples. Proteomics, 14 (9). pp. 1048-1052.

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Official URL: http://edoc.unibas.ch/dok/A6308363

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Abstract

AMPylation is a posttranslational modification (PTM) that has recently caught much attention in the context of bacterial infections as pathogens were shown to secrete Fic proteins that AMPylate Rho GTPases and thus interfere with host cell signaling processes. Although Fic proteins are widespread and found in all kingdoms of life, only a small number of AMPylation targets are known to date. A major obstacle to target identification is the limited availability of generic strategies allowing sensitive and robust identification of AMPylation events. Here, we present an unbiased MS-based approach utilizing stable isotope-labeled ATP. The ATP isotopes are transferred onto target proteins in crude cell lysates by in vitro AMPylation introducing specific reporter ion clusters that allow detection of AMPylated peptides in complex biological samples by MS analysis. Applying this strategy on the secreted Fic protein Bep2 of Bartonella rochalimae, we identified the filamenting protein vimentin as an AMPylation target that was confirmed by independent assays. Vimentin represents a new class of target proteins and its identification emphasizes our method as a valuable tool to systematically uncover AMPylation targets. Furthermore, the approach can be generically adapted to study targets of other PTMs that allow incorporation of isotopically labeled substrates.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Infection Biology > Molecular Microbiology (Dehio)
05 Faculty of Science > Departement Biozentrum > Services Biozentrum > Proteomics (Schmidt)
05 Faculty of Science > Departement Biozentrum > Infection Biology > Molecular Microbiology (Jenal) > Molecular Microbiology (Harms)
05 Faculty of Science > Departement Biozentrum > Growth & Development > Molecular Microbiology (Jenal) > Molecular Microbiology (Harms)
UniBasel Contributors:Dehio, Christoph and Glatter, Timo and Harms, Alexander and Pieles, Kathrin and Schmidt, Alexander and Schmidt, Alexander
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:Wiley-VCH Verlag
ISSN:1615-9853
e-ISSN:1615-9861
Note:Publication type according to Uni Basel Research Database: Journal article
Language:English
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Last Modified:19 Aug 2020 10:31
Deposited On:07 Nov 2014 08:29

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