Assessing Metabolite Interactions With Chloroplastic Proteins via the PISA Assay.

Plants rely on metabolite regulation of proteins to control their metabolism and adapt to environmental changes, but studying these complex interaction networks remains challenging. The proteome integral solubility alteration (PISA) assay, a high-throughput chemoproteomic technique, was originally developed for mammalian systems to investigate drug targets. PISA detects changes in protein stability upon interaction with small molecules, quantified through LC-MS. Here, we present an adapted PISA protocol for chloroplasts to identify potential protein interactions with ascorbate. Chloroplasts are extracted using a linear Percoll gradient, treated with multiple ascorbate concentrations, and subjected to heat-induced protein denaturation. Soluble proteins are extracted via ultracentrifugation, and proteome-wide stability changes are quantified using multiplexed LC-MS. We provide instructions for deconvolution of LC-MS spectra and statistical analysis using freely available software. This protocol enables unbiased screening of protein regulation by small molecules in plants without requiring prior knowledge of interaction partners, chemical probe design, or genetic modifications. Key features • Optimization of the PISA assay to study protein-ligand interactions in plant chloroplasts, including isolation of chloroplasts. • Study of regulation on a proteome level, without genetic manipulation or prior knowledge of interaction partners. • High proteome coverage, low sample requirement, 5-fold reduction of TMT-labeling cost, and short LC-MS analysis time. • Adaptable to other organisms, such as bacteria, with minor modifications.