Rapid Identification of Protein-Protein Interactions in Plants

Youjun Zhang, Roberto Natale, Adilson Pereira Domingues, Mitchell Rey Toleco, Beata Siemiatkowska, Norma Fàbregas, Alisdair R. Fernie

Producció científica: Article en revista indexadaArticleAvaluat per experts

21 Cites (Scopus)

Resum

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).

Idioma originalAnglès
Pàgines (de-a)e20099
RevistaCurrent protocols in plant biology
Volum4
Número4
DOIs
Estat de la publicacióPublicada - 1 de des. 2019
Publicat externament

Fingerprint

Navegar pels temes de recerca de 'Rapid Identification of Protein-Protein Interactions in Plants'. Junts formen un fingerprint únic.

Com citar-ho