In planta chemical crosslinking and mass spectrometry analysis of protein structure and interaction in Arabidopsis

Mass spectrometry analysis in combination with the site-specific chemical cross-linking has emerged as a powerful method in study of three-dimensional structure of protein complex and in mapping of protein-protein interactions (PPIs). Even though in vitro cross-linking experiments have been widely applied to investigate the specific interactions of a bait protein and its targets, the measurement of in vivo protein tertiary structure and PPIs has been problematic and strenuous due to the dynamic nature of the biological systems and a lower number of cross-linked peptides that can be isolated via MudPIT (Multidimensional Protein Identification Technology) for mass spectrometry analysis. Using Arabidopsis thaliana as a model multicellular eukaryotic organism, we have attempted to develop an improved in vivo chemical cross-linking and mass spectrometry (or IPXL-MS) workflow, which aims at optimizing the in vivo cross-linking conditions, establishing of a MudPIT procedure for enrichment of cross-linked peptides, and developing an integrated software program to identify the in planta chemical cross-linked peptides, by which three pairs of in vivo cross-linked peptides of high-confidence has been identified twice from two independent biological replicates. This work has demarked a beginning of alternative proteomic approach in study of in vivo protein tertiary structure and PPIs in higher plants. This in vivo cross-linking approach may be applied into other model multicellular organisms, such as mouse, for molecular systems biological research.