Fast hybrid mass spectrometer for complexome analyses

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 543577782

The requested timsTOF HT mass spectrometer will replace the LTQ Orbitrap XL mass spectrometer that has so far been used by the applicant and will be shut down for economic and technical reasons. The Institute for Physiology II investigates the molecular basis of fast signal transduction at and across the plasma membrane, in particular of neurons in the mammalian brain. A central organization principle behind the exquisite spatial and temporal precision of this signal transduction is the assembly of proteins into macro-molecular complexes and networks. For unraveling their molecular appearance and composition, the institute has developed several approaches of (functional) proteomics over the last two decades and successfully applied them to a series of ion channels, membrane receptors and transporters. The experimental workflows comprise (i) isolation of membrane fractions from native tissues, (ii) affinity-purification of target proteins (multi-epitope AP-MS, "meAP-MS"), (iii) highly resolved separation of protein fractions and membrane sub-fractionations (complexome analysis or organellar proteomics), and (iv) high-resolution nano-HPLC coupled mass spectrometry for identification and quantification of proteins including their post-translational modifications like phosphorylation, alkylation or lipid modifications. Accordingly, their analysis requires high-performance mass spectrometry with respect to mass resolution (mass/charge), sensitivity, dynamic range and scan/cycle speed, in particular for high-resolution complexome analyses (by the ‘cryo-slicing blue native MS‘, csBN-MS technique, recently developed at the institute), which require extended measurement series (200-400 measurements per biological sample) with high analytical coverage. The latter will be run on the requested instrument which - due to its very high speed (>5fold with still superior performance in direct comparison with the presently available instruments) and quantification over an extended dynamic range (i.e. obtaining quantitative data for a larger number of proteins/peptides covering 4-5 orders of magnitude within an individual measurement) - is ideally suited. Such high-resolution complexome analyses enable for the first time a comprehensive view on the molecular organization and dynamics of protein complexes in different biological systems. Acquisition of the timsTOF HT is, therefore, of key importance for the future work/projects of the applicant's group.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Schnelles Hybrid-Massenspektrometer für Komplexom-Analysen
• Instrumentation Group: 1700 Massenspektrometer
• Applicant Institution: Albert-Ludwigs-Universität Freiburg
• Leader: Professor Dr. Bernd Fakler