DFG Major Research Instrumentation: HPLC/ESI Ion Trap Mass Spectrometer

Project: DFG ProjectsDFG Major Research Instrumentation

Project Details

Description

The device is used for protein measurements to determine the crystal structure of proteins.
These proteins may need to be modified. One type of modification is the incorporation of selenomethionine instead of the naturally occurring amino acid methionine into the protein to be analysed. In order to measure the degree of incorporation of selenomethionine, various test proteins (cytochrome c, ovalbumin, lysozyme, clostripain, carbonic anhydrase) were initially measured to establish the measurement procedure. From the measurements of the natural and the selenomethionine-modified protein HtrA, an oligomeric serine protease, the incorporation of 7 selenomethionine residues into the protein could be determined. Since the protein sequence contains only 7 methionine residues, a complete incorporation of selenomethionine residues could be confirmed. The molar mass of other proteins being worked on at the institute, such as a protease from the St. Louis encephalitis virus (SLEV), was also routinely determined.

Inhibitors: Structure-based antiviral lead compounds are routinely analysed with the ESI-MS device with regard to their molecular size. The measurements are usually carried out using the positive-mode method, for some compounds also using the negative-mode method. In most cases, MS/MS spectra of the compounds are also measured to determine the structure more precisely. The target molecules against which these lead compounds are directed are essentially non-structural proteins (Nsps) from pathogenic RNA viruses such as SARS-CoV, West Nile virus, Chikungunya virus, Coxsackievirus and other enteroviruses. The 3C proteases from these viruses are used as the main target proteins. The synthesised lead compounds belong chemically to the group of Michael acceptors, benzotriazol esters, peptide aldehydes, benzanilides and substituted pyrroles. A total of more than 300 compounds and their precursors were measured. Of these, around 120 substances are intended for patenting.

Nano-LC measurements: Tryptic digested proteins were separated using nano-HPLC and analysed using the ESI-MS device. The samples analysed were mainly proteins that play a role in the infection or inflammation process, such as RelA (which plays a role in the adaptation of the prokaryotic metabolism and its gene expression to environmental changes and acts as a virulence factor), CPAF (a chlamydial protease), two fragments of DegQ (a chaperone protease from the periplasm of Legionella micdadei), PfICP (a cysteine protease inhibitor from Plasmodium falciparum) and the C- and N-terminal domain of the Mip protein (macrophage infectivity potentiator, an essential virulence factor) from Legionella and Chlamydia. As these proteins are partially unstable, in addition to the native proteins, degradation products were also investigated in order to characterise the degradation more precisely (C-terminal, N-terminal or interfaces within the protein). Furthermore, domain boundaries were also determined using ESI-MS. In some cases, the sequence coverage reached values of over 60%, which enabled unambiguous identification.

In collaboration with the Institute of Chemistry, the molecular weight of the peracetylated sugar 2-deoxy-2-N-13C-acetyl- 1,3,4,6-tetra-O-acetyl-ß-D glucopyranose and its precursors was analysed. Furthermore, investigations were carried out on the dimerisation of the galactosyltransferases GTA and GTB and the size of domain A2 of the von Willebrand factor was determined in collaboration with Prof. Karsten Seeger.

Department of Dermatology and Venereology: In collaboration with Prof Ralf Ludwig, the size of proteins involved in immunomodulation was measured. i

Institute of Physics: In collaboration with Prof. Dr Christian Hübner, the size of various fluorescence-labelled proteins was determined.

Institute of Medical Microbiology and Hygiene: In collaboration with Prof. Dr Jan Rupp, an attempt was made to identify proteins in the inclusion membrane of chlamydiae.

StatusActive
Effective start/end date01.01.07 → …

UN Sustainable Development Goals

In 2015, UN member states agreed to 17 global Sustainable Development Goals (SDGs) to end poverty, protect the planet and ensure prosperity for all. This project contributes towards the following SDG(s):

  • SDG 3 - Good Health and Well-being

Research Areas and Centers

  • Academic Focus: Center for Infection and Inflammation Research (ZIEL)

DFG Research Classification Scheme

  • 204-04 Virology
  • 201-01 Biochemistry

Funding Institution

  • DFG: German Research Association