. . . at the DFG core facility
If you are interested in performing HDX-MS experiments, please contact our staff scientist Dr. Wieland Steinchen (Wieland.email@example.com). He will get in contact with you to discuss and plan your HDX-MS experiment. The DFG core facility offers three standardized workflows covering the most requested HDX-MS experiments:
Determination of conformational dynamics and protein folding.
This workflow allows determining the conformational dynamics and flexibilities in proteins, their folding intermediates and complexes. Different conditions, such as ionic strength, pH, temperature or light can be investigated.
Determination of protein-ligand interface.
This workflow allows mapping of interfaces required for the interaction of a protein with its ligand (e.g. another protein, RNA, DNA, lipid and small molecules). Ligand-induced conformational rearrangements in proteins can be determined. It also allows delineating the architecture of macromolecular assemblies containing multiple protein subunits.
Construct optimization for protein crystallization.
Highly flexible regions within proteins can hinder their crystallization. HDX-MS allows the identification of such regions and therefore can be used to generate protein variants with better propensity to crystallize. Thereby, HDX-MS can be beneficial for generating high-quality crystals suitable for X-ray crystallography.
If you require a customized experiment, which is not described in the standardized workflows, please get in touch with us.
Sample preparation and size
The user provides the samples and additives (e.g. ligands) for HDX-MS experiments. Samples should be delivered deep-frozen. In case the protein cannot be deep-frozen, please contact the staff scientist. For each protein state (e.g. apo- or ligand-bound), 200 µl of 50 µM protein are ideal. If you are unable to provide this amount, please contact the staff scientist. Proteins or protein complexes should ideally be purified by size-exclusion chromatography.
Data processing and evaluation
HDX-MS experiments provide huge amounts of raw data that as such cannot easily be interpreted by untrained users. Therefore, our staff will process the raw data to extract the information relevant for the project that has been defined together with the user. We have installed three workstations with all software required for HDX-MS data analysis. You and your co-workers are welcome to use them.
Use of any HDX-MS data generated by the DFG core facility in publications absolutely requires an acknowledgement stating: “We acknowledge support from the “DFG-core facility for interactions, dynamics and macromolecular assembly structure” at the Philipps-University Marburg.” If any result produced within the facility that involved an intellectual or scientific contribution of a staff member, will be published, this staff member should appear as a co-author of the publication.
The users will get structural and evaluated HDX-data electronically and, on demand, also the complete set of raw data (which usually is ~100 GB or even more). All raw data will be stored electronically within the facility on a local storage within the facility for at least three years. Furthermore, a data backup will be stored on a tape drive operated by the computing centre of the Philipp-University Marburg for additional ten years so that we will guarantee at least 13 years of raw data availability.
Prices are available on request and will be discussed prior to the beginning of your experiment.