Assessing Metabolite Interactions With Chloroplastic Proteins via the PISA Assay

Karlsson, Anna and Sporre, Emil and Strandberg, Linnéa and Tóth, Szilvia Zita and Hudson, Elton (2025) Assessing Metabolite Interactions With Chloroplastic Proteins via the PISA Assay. BIO-PROTOCOL, 15 (9). No. e5298. ISSN 2331-8325

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Abstract

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 Arabidopsis thaliana 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.

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