Session: Parallel session 2 - Interactomics

Assessment of Mitochondrial Inhibitor Pesticides-Protein Interactions in Liver cells using Limited Proteolysis coupled to mass spectrometry

Natalia PINTO DE ALMEIDA¹, Sarah ALILAT¹, Pierre-Jean FERRON¹, Valérie FESSARD¹, Thibaut LÉGER¹

¹ANSES, French Agency for Food, Environmental and Occupational Health & Safety, Toxicology of Contaminants Unit, Fougères Laboratory, Fougères , France

Introduction: Pesticides are defined as chemicals used to eliminate pests like insects, rodents, fungi, and plants. Many pesticides are mitochondrial inhibitors linked to neurodegenerative diseases, including Parkinson's. These compounds are absorbed by hepatocytes, where they are metabolized and potentially induce toxic effects. Toxicity is mainly due to interactions between chemicals and proteins, disrupting biological processes. This study aims to explore the interaction mitochondrial inhibitor pesticides-proteins to understand their mechanisms of action and liver toxicity.

Methodology: We used the Limited Proteolysis coupled to mass spectrometry (LIP-MS) protocol to assess chemical-protein interactions. Differentiated HepaRG cells underwent protein extraction in native conditions (Pierce™ IP Lysis Buffer). The lysate was incubated with tebufenpyrad, a complex I inhibitor, (TEBU, 10µM, 5min) and subjected to limited proteolysis (LP) with Pronase (PRO) (1:100) at different reaction times (5, 15, and 30min). Samples were then diluted in SDS solution for tryptic digestion using the STRAP (PROTIFI) protocol. Analysis was conducted using a Tims TOF Pro 2 (Bruker) mass spectrometer coupled to a nanoHPLC.

Results: Preliminary results identified around 4,200 proteins and 37,000 peptides in DDA analysis. Increasing the LP duration with PRO showed a trend of decreasing protein and peptide identification, though not statistically significant, and no change in the percentage of half tryptic peptides (~18%) and missed cleavages (~23%). Proteins STAT2/3, ATP5B/A1 were found as potential interactors with TEBU, although further evaluation and confirmation are needed.

Conclusion: We showed that 5min of LP with PRO is sufficient for good proteome coverage and high number of LIP sites. Future tests will explore various modifications (chemical concentrations, lysis buffers, DIA mode analysis) and the analysis of other mitochondrial Inhibitor pesticides.