Integrated Proteomics and Lipidomics Platform

• Applicants: Laura Bindila, Ph.D.; Dr. Ute Distler
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2021 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 446605368

The overarching goal of the SPP 2225 is to elucidate the evolved exit pathways employed by bacterial, parasitic and fungal pathogens with relevance for human health. The integrated proteomics and lipidomics platform will provide the SPP with high-end mass spectrometry technology for quantitative proteomic and lipidomic analysis and bioinformatics expertise, enabling extensive molecular output in various infection models. We will tailor and implement sample preparation and analytical protocols for specific cells, pathogens and intracellular organelles. In this program, we will provide quantitative datasets with deep proteome and lipidome coverage, which will allow the SPP participants to dissect the mechanisms that trigger, regulate, and synchronize the exit events at proteome and lipidome level. Subproject 1 (SP1 - Distler) provides state-of-the-art technologies and infrastructure to conduct high-throughput quantitative proteomics. Integrating lab and computational platforms at the Institute of Immunology, SP1 will offer the SPP full proteomic solutions. These are comprised of optimized and standardized protocols for mid- to high-throughput sample preparation, the generation of high-quality label-free quantitative proteomics datasets, access to dedicated analysis tools and the required expertise for proteomics data evaluation. SP1 is equipped with six latest generation, high-resolution mass spectrometry (MS) platforms including two Bruker timsTOF-Pro2, one timsTOF-HT, one timsTOF SCP and two Thermo Fisher Scientific Orbitrap Exploris 480 instruments. Applying high-resolution MS-based quantitative proteomics and bioinformatics solutions, SP1 will help the SPP to characterize and understand the dynamics of proteome organization upon infection and pathogen egress. Moreover, SP1 will enable the SPP to monitor changes of post-translational modifications, such as phosphorylation, induced by pathogen exit strategies as well as to detect key proteins involved in host-pathogen interactions. This will define major molecular mediators and effectors required for pathogen egress. Subproject 2 (SP2 - Bindila) provides new generation analytical platforms for unbiased and targeted lipidomics. Two high-end instruments, timsTOF Pro and timsTOF flex, along with software solutions and expertise for lipid identification and quantification are available for untargeted lipidomics. 4 Dimensional (4D)-lipidomics using liquid chromatography /trapped ion mobility mass spectrometry (LC/tims) and 3D lipidomics using matrix-assisted laser desorption (MALDI) MS allow comprehensive characterization of lipidome changes in hosts, pathogens and organelles upon infection and egress. Multiplexed targeted assays using LC- multiple reaction monitoring (MRM) on QTRAP instrument facilitates investigation of low abundant lipid mediators of the pathogens' infection and egress mechanisms. In the phase II, we will also implement a dual proteome/lipidome protocol from the same sample.

• DFG Programme: Priority Programmes
• Subproject of: SPP 2225: Exit strategies of intracellular pathogens