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 d...
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| Format: | Article |
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Bio-protocol LLC
2025-05-01
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| Series: | Bio-Protocol |
| Online Access: | https://bio-protocol.org/en/bpdetail?id=5298&type=0 |
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| author | Anna Karlsson Emil Sporre Linnéa Strandberg Szilvia Tóth Elton Hudson |
| author_facet | Anna Karlsson Emil Sporre Linnéa Strandberg Szilvia Tóth Elton Hudson |
| author_sort | Anna Karlsson |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-abf9f339d83545f29c14b167ac6f7464 |
| institution | OA Journals |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-abf9f339d83545f29c14b167ac6f74642025-08-20T02:14:24ZengBio-protocol LLCBio-Protocol2331-83252025-05-0115910.21769/BioProtoc.5298Assessing Metabolite Interactions With Chloroplastic Proteins via the PISA AssayAnna Karlsson0Emil Sporre1Linnéa Strandberg2Szilvia Tóth3Elton Hudson4School of Engineering Science in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, SwedenSchool of Engineering Science in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, SwedenInstitute for Integrative Biology of the Cell, Université Paris-Saclay, Paris, FranceSchool of Engineering Science in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, SwedenSchool of Engineering Science in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, SwedenPlants 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.https://bio-protocol.org/en/bpdetail?id=5298&type=0 |
| spellingShingle | Anna Karlsson Emil Sporre Linnéa Strandberg Szilvia Tóth Elton Hudson Assessing Metabolite Interactions With Chloroplastic Proteins via the PISA Assay Bio-Protocol |
| title | Assessing Metabolite Interactions With Chloroplastic Proteins via the PISA Assay |
| title_full | Assessing Metabolite Interactions With Chloroplastic Proteins via the PISA Assay |
| title_fullStr | Assessing Metabolite Interactions With Chloroplastic Proteins via the PISA Assay |
| title_full_unstemmed | Assessing Metabolite Interactions With Chloroplastic Proteins via the PISA Assay |
| title_short | Assessing Metabolite Interactions With Chloroplastic Proteins via the PISA Assay |
| title_sort | assessing metabolite interactions with chloroplastic proteins via the pisa assay |
| url | https://bio-protocol.org/en/bpdetail?id=5298&type=0 |
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