High-content Proteomics

Over the past five years, we have made a number of advances in the precise, high-throughput and large-scale characterization of proteomes, thanks to specific actions on the biochemical, the analytical and the data processing fronts. These achievements are instrumental to implement the project of the team.

Research focus

Automation of biochemical preparation of samples

PepS: a microfluidic device for plasma proteome preparation
In collaboration with CEA-Leti, we developed a microfluidic device allowing bedside whole blood processing for MSbased analysis of plasma proteome. We were able to assess the performance and repeatability of PepS for plasma processing and compared it to manual preparation using discovery and targeted proteomic analyses (Gilquin et al., 2021, Anal Chem).

Development of specific protocols on an automated liquid handling workstation
In the context of a proteomics project, it is essential to adapt the biochemical preparation according to the biomedical question posed on the one hand and the constraints of the analytical sciences on the other. The team’s long-term experience in this field has led us to select a limited number of protocols for which automation would be relevant in order to improve reproducibility and throughput for large series of samples. For this we a have acquired a Fluent workstation and we are currently developing several protocols, notably dedicated to phospho-enrichment (for phosphoproteomics and crosslinking-MS purposes) and total proteome preparation (in-solution and on-beads – SP3 – trypsin digestion)

High-throughput MS-based measurements

nanoLC-MS/MS analysis methods for large-scale studies in “discovery” mode, were further developed notably with the addition of a novel additional ion fractionation method (ion mobility) on faster and ever
more sensitive instruments. Together with the implementation of highly efficient chromatography separation of peptides, it allowed us to significantly improve the depth of analysis of the proteomes studied, making it possible, for example, to exceed the 10,000 phosphorylation sites and 8,000 proteins identified and quantified in a single project (Al Tarrass M et al., 2024, Cell Commun Signal; Pastor F et al., 2024, Front Microbiol; Antunes AV et al., 2024, Nature). In the near future, we will be pushing back the limits of sensitivity by developing innovative acquisition strategies on cutting-edge MS instruments, while accelerating the throughput of our analyses using state-of-the-art chromatographic systems in order to be able to analyze several dozen samples containing very small amounts of
proteins per day.

Structural proteomics

we develop and apply cutting-edge structural proteomics strategies to investigate the organization and
dynamics of protein complexes and interaction networks. The systems we address range from purified assemblies to functional photosynthetic membranes, and even intact living cells. Our approaches are specifically designed to capture structural information under physiologically relevant conditions, bridging molecular resolution with functional context. By combining in vivo crosslinking, quantitative mass spectrometry, and integrative data analysis, we dissect how macromolecular assemblies remodel in response to environmental or cellular cues. A strong emphasis is placed on workflow optimization to deliver high-quality, information-rich datasets while minimizing instrument time and sample consumption. These methodologies are currently deployed in a variety of projects, including the investigation of rapid
structural adaptations of photosynthetic and respiratory machineries in plants and microalgae (in close collaboration with LPCV), as well as the characterization of protein complexes in partnership with a selected set of collaborators.

Development of computational methods and tools

In the proteomics fields, experimental design, sample preparation, data acquisition and data analysis must be considered of equal importance. As a result, being able to control the way in which data are processed, analyzed and interpreted is crucial. To enable this control, we choose to develop our own methodologies and software tools at key stages of the process. These developments ranges from tools to manage data generated by the MS instrumentation to environments dedicated to the interpretation of the proteomics results.