An in planta chemical cross-linking-based quantitative interactomics (IPQCX-MS) workflow has been developed to investigate in vivo protein-protein interactions and alteration in protein structures in a model organism, Arabidopsis thaliana. A chemical cross-linker, azide-tag-modified disuccinimidyl pimelate (AMDSP), was directly applied onto Arabidopsis tissues. Peptides produced from protein fractions of CsCl density gradient centrifugation were dimethyl-labeled, from which the AMDSP cross-linked peptides were fractionated on chromatography, enriched, and analyzed by mass spectrometry. ECL2 and SQUA-D software were used to identify and quantitate these cross-linked peptides, respectively. These computer programs integrate peptide identification with quantitation and statistical evaluation. This workflow eventually identified 354 unique cross-linked peptides, including 61 and 293 inter- and intraprotein cross-linked peptides, respectively, demonstrating that it is able to in vivo identify hundreds of cross-linked peptides at an organismal level by overcoming the difficulties caused by multiple cellular structures and complex secondary metabolites of plants. Coimmunoprecipitation and super-resolution microscopy studies have confirmed the PHB3-PHB6 protein interaction found by IPQCX-MS. The quantitative interactomics also found hormone-induced structural changes of SBPase and other proteins. This mass-spectrometry-based interactomics will be useful in the study of in vivo protein-protein interaction networks in agricultural crops and plant-microbe interactions.
Keywords: 4C proteomic workflow; Arabidopsis thaliana; ECL2; IPQCX−MS; in planta quantitative interactomics; in vivo cross-linking.