Assessing Metabolite Interactions With Chloroplastic Proteins via the PISA Assay
Year: 2025
Type: article
Abstract: 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.
Source: BIO-PROTOCOL
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: diamond
OpenAlex ID: W4409513194