ausblenden:
Schlagwörter:
affinity purification mass spectrometry, fold change abundance, protein-protein interaction
Zusammenfassung:
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
