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  Rapid Identification of Protein-Protein Interactions in Plants

Zhang, Y., Natale, R., Domingues Júnior, A., Toleco, M., Siemiatkowska, B., Fàbregas, N., et al. (2019). Rapid Identification of Protein-Protein Interactions in Plants. Current Protocols in Plant Biology, 4(4): e20099. doi:10.1002/cppb.20099.

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Zhang, YJ1, Author              
Natale, R.1, Author              
Domingues Júnior, A.P.1, Author              
Toleco, M.R.1, Author              
Siemiatkowska, B.1, Author              
Fàbregas, Norma1, Author              
Fernie, A. R.1, Author              
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1Central Metabolism, Department Willmitzer, Max Planck Institute of Molecular Plant Physiology, Max Planck Society, ou_1753339              

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Free keywords: affinity purification mass spectrometry, fold change abundance, protein-protein interaction
 Abstract: Abstract Enzyme-enzyme interactions can be discovered by affinity purification mass spectrometry (AP-MS) under in vivo conditions. Tagged enzymes can either be transiently transformed into plant leaves or stably transformed into plant cells prior to AP-MS. The success of AP-MS depends on the levels and stability of the bait protein, the stability of the protein-protein interactions, and the efficiency of trypsin digestion and recovery of tryptic peptides for MS analysis. Unlike in-gel-digestion AP-MS, in which the gel is cut into pieces for several independent trypsin digestions, we uses a proteomics-based in-solution digestion method to directly digest the proteins on the beads following affinity purification. Thus, a single replicate within an AP-MS experiment constitutes a single sample for LC-MS measurement. In subsequent data analysis, normalized signal intensities can be processed to determine fold-change abundance (FC-A) scores by use of the SAINT algorithm embedded within the CRAPome software. Following analysis of co-sublocalization of “bait” and “prey,” we suggest considering only the protein pairs for which the intensities were more than 2% compared with the bait, corresponding to FC-A values of at least four within-biological replicates, which we recommend as minimum. If the procedure is faithfully followed, experimental assessment of enzyme-enzyme interactions can be carried out in Arabidopsis within 3 weeks (transient expression) or 5 weeks (stable expression). © 2019 The Authors. Basic Protocol 1: Gene cloning to the destination vectors Alternate Protocol: In-Fusion or Gibson gene cloning protocol Basic Protocol 2: Transformation of baits into the plant cell culture or plant leaf Basic Protocol 3: Affinity purification of protein complexes Basic Protocol 4: On-bead trypsin/LysC digestion and C18 column peptide desalting and concentration Basic Protocol 5: Data analysis and quality control

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Language(s): eng - English
 Dates: 2019
 Publication Status: Published in print
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 Rev. Type: -
 Identifiers: DOI: 10.1002/cppb.20099
BibTex Citekey: doi:10.1002/cppb.20099
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Title: Current Protocols in Plant Biology
Source Genre: Journal
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Pages: - Volume / Issue: 4 (4) Sequence Number: e20099 Start / End Page: - Identifier: -