• Location: PAPPSO, UMR Génétique Quantitative et Evolution - Le Moulon, Gif-sur-Yvette (France)
  • Date: 1st semester 2025

Context: Phosphorylation is one of the most important post-translational modifications (PTMs) of proteins, affecting about one third of all proteins, and plays a key role in regulating many important biological processes. However, like any other PTM, phosphorylation cannot be predicted from the genome or transcriptome, and its study requires specific techniques. Phosphoproteomics is currently the main approach to characterize phosphoproteins, but, as compared to proteomics, it requires special considerations in sample handling, data acquisition, and post-acquisition processing. The PAPPSO platform routinely performs phosphoproteomic analyses using a workflow based on i) protein extraction and digestion, ii) stable isotope labeling of peptides to allow sample multiplexing and improved quantitative comparisons, iii) sample fractionation for a deeper analysis where necessary, iv) phosphopeptide enrichment. While this workflow provides broad phosphoproteome coverage, it remains complex and time consuming, limiting its use in large scale studies. To address this issue, we are exploring two avenues for improving the workflow to make it faster and more efficient:

  • shifting from individual tubes to a plate-based format, which is more suitable for high-throughput sample processing,
  • operating the mass spectrometer in the data independent acquisition (DIA) mode instead of the classically used data dependent acquisition (DDA) mode. DIA differs from DDA mainly in the way peptide ions are isolated for fragmentation in the mass spectrometer. This offers better proteome coverage, potentially eliminating the need for sample fractionation.

Objective: The goal of thi internship is to optimise the phosphoproteomics workflow for handling large plant and bacterial sample cohorts. The project will focus on three key aspects:

  • Validating labeling efficiency, enrichment rate, and repeatability when processing samples in plate,
  • Developping and implementing of a DIA approach tailored for multiplexed samples,
  • Based on the results, fine-tuning a DIA method optimized specifically for phosphopeptide analysis.

Application: Please send a CV and motivation letter to Mélisande Blein-Nicolas (melisande.blein-nicolas@inrae.fr, 01 69 15 68 06).