Abstract
The automated SPOT (synthetic peptide arrays on membrane support technique) synthesis technology has entrenched as a rapid and robust method to generate peptide libraries on cellulose membrane supports. The synthesis method is based on conventional Fmoc chemistry building up peptides with free N-terminal amino acids starting at their cellulose-coupled C-termini. Several hundreds of peptide sequences can be assembled with this technique on one membrane comprising a strong binding potential due to high local peptide concentrations. Peptide orientation on SPOT membranes qualifies this array type for assaying substrate specificities of N-recognins, the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Pioneer studies described binding capability of mammalian and yeast enzymes depending on a peptide’s N-terminus. SPOT arrays have been successfully used to describe substrate specificity of N-recognins which are the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Here, we describe the implementation of SPOT binding assays with focus on the identification of N-recognin substrates, applicable also for plant NERD enzymes.
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Acknowledgements
We thank Christian Behn from INTAVIS for personal advices on the ResPep SL SPOT peptide synthesis and we are grateful to Petra Majovsky and Wolfgang Hoehenwarter for constant support in mass spectrometry and proteome analytics. This work was supported by a grant for setting up the junior research group of the ScienceCampus Halle—Plant-based Bioeconomy to N.D., a Ph.D. fellowship of the ScienceCampus Halle to M.K. Financial support came from the Leibniz Association, the state of Saxony-Anhalt, the Deutsche Forschungsgemeinschaft (DFG) Graduate Training Center GRK1026 “Conformational Transitions in Macromolecular Interactions” at Halle, and the Leibniz Institute of Plant Biochemistry (IPB) at Halle, Germany.
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Klecker, M., Dissmeyer, N. (2016). Peptide Arrays for Binding Studies of E3 Ubiquitin Ligases. In: Lois, L., Matthiesen, R. (eds) Plant Proteostasis. Methods in Molecular Biology, vol 1450. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3759-2_7
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DOI: https://doi.org/10.1007/978-1-4939-3759-2_7
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