NanoLC-ESI-MSMS for bottom-up proteomics

• Subject Area: Microbiology, Virology and Immunology
• Term: Funded in 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 564838960

Proteomics is a very broad field and one of the main pillars of systems biology. We are using proteomics mainly for the systemic understanding of host-pathogen interactions in bacterial and viral infections and for the development of new therapeutic approaches for the treatment of multi-drug resistant pathogens, in particular for the identification of targets for novel anti-infectives and for antibody-based approaches. The focus is on bottom-up proteomics approaches for the identification and relative quantification of proteins (label-free, SILAC, TMT) in highly complex protein samples. Special attention is given to the identification and quantification of small proteins and proteoforms. As these can often only be identified from a single unique peptide, high-resolution MS2 spectra, high mass accuracy and long, uninterrupted fragment ion series are required to enable de novo sequencing of these peptides. Other important techniques include the analysis of post-translational modifications (e.g. phosphorylation), the characterisation of specific protein complexes that are formed during infection using cross-linking MS, the interaction of proteins with metabolites and proteins using thermal proteome profiling, and the fine mapping of epitopes for specific therpeutic antibodies. For all these applications, a combination of high-resolution nano liquid chromatography coupled to a mass spectrometer with high sensitivity (< 1 pg), high resolution and mass accuracy (≥ 50,000; < 5 ppm), high quality MS2 spectra with a scan rate of at least 10 Hz and a wide dy-namic measurement range (at least three orders of magnitude) is required. In addition, DDA data acquisition is necessary to unambiguously assign spectra to individual peptides and to proteogenomically prove the existence of small proteins not yet included in genome annotations. For highly complex in-depth proteomic (e.g. metaproteomic) analyses, a large capacity for MS2 spectra is advantageous. The existing equipment, consisting of a nanoAQuity UPLC system and an LTQ Orbitrap Velos pro mass spectrometer, which was installed more than 13 years ago, no longer meets these requirements and is to be replaced by a new nanoLC-ESI-MSMS system. The combination of nano-liquid chromatography, electrospray ionisation and tandem mass spectrometry applied here is extremely useful and powerful for investigating the molecular processes at the proteome level that enable microorganisms to successfully adapt to the host environment, modulate or protect themselves from the host immune system and successfully resist antibiotics. The data obtained will significantly improve our understanding of the virulence and resistance mechanisms of important pathogens, which is crucial for the development of new diagnostic and therapeutic approaches.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: NanoLC-ESI-MSMS für Bottom-up Proteomik
• Instrumentation Group: 1700 Massenspektrometer
• Applicant Institution: Technische Universität Braunschweig
• Leader: Professorin Dr. Susanne Engelmann