Identifying and understanding protein-protein and protein-ligand interactions are a critical part of the development of drug candidates, biotherapeutics and agrochemicals. Therefore, demand for techniques which can map these interactions is rapidly increasing.
The University of Nottingham Protein Footprinting Service uses cutting-edge Carbene Footprinting technology, developed by Professor Neil Oldham, to provide a high quality and cost-effective way to gather critical data for your biological products and their interactions.
We use a bespoke diazirine-based label, which upon irradiation with near-UV light undergoes photolysis to yield a carbene, which subsequently irreversibly reacts with the target protein on a nanosecond timescale. The reaction between the label and the accessible protein surface causes an increase in mass, which can be observed by nanoLC-MS/MS analysis via a bottom-up proteomic workflow. Labelling of the protein with the chemical reagent is performed in the presence and absence of a binding partner, forming a differential study. The presence of the binding partner will shield a region of the protein’s surface from the label, and this data is compared to that of the unbound protein to identify the binding site.
Left: Carbenes are able to react with the entire surface of unbound Lysozyme (blue)
Right: The carbenes cannot access the binding site between Lysozyme (blue) and NAG5 (pink)
Hydrogen-Deuterium Exchange (HDX) and Hydroxy Radical Protein Footprinting (HRPF, FPOP) are other examples of footprinting commonly used, although both techniques have limitations associated with them. Carbene Footprinting offers several advantages over these techniques, including:
- The labelling reaction is irreversible.
- Labelling causes a large detectable mass shift.
- Probe is stable in ambient light and in solution until photochemical activation.
As part of our Protein Footprinting Service, we offer bespoke agreements for all our clients, ensuring the exact needs of your study are met. We adopt a multi-stage approach to your project, allowing you to evaluate results at every stage of analysis.
Our current services include, but are not limited to:
- Protein-Protein Interaction Mapping
- Protein-Ligand Interaction Mapping
- Antibody-Antigen Epitope/Paratope Mapping
- Membrane Protein Interaction Studies
Our team
Enquiries
If you are interested in hearing more about our Protein Footprinting service, how it may benefit your application or obtaining a quote for your project, please don’t hesitate to contact us by submitting an enquiry below:
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If you would like to read more about the applications of our carbene footprinting work, please see some of our publications below:
- Manzi L, Barrow AS, Scott D, et al. Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions. Nat Commun. 2016;7. doi:10.1038/ncomms13288
- Manzi L, Barrow AS, Hopper JTS, et al. Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane-Spanning Protein. Angewandte Chemie - International Edition. 2017;56(47):14873-14877. doi:10.1002/anie.201708254
- Jenner M, Kosol S, Griffiths D, et al. Mechanism of intersubunit ketosynthase-dehydratase interaction in polyketide synthases. Nat Chem Biol. 2018;14(3):270-275. doi:10.1038/nchembio.2549
- Bellamy-Carter J, Oldham NJ. PepFoot: A Software Package for Semiautomated Processing of Protein Footprinting Data. J Proteome Res. 2019;18(7):2925-2930. doi:10.1021/acs.jproteome.9b00238
- Kosol S, Gallo A, Griffiths D, et al. Structural basis for chain release from the enacyloxin polyketide synthase. Nat Chem. 2019;11(10):913-923. doi:10.1038/s41557-019-0335-5
- Lloyd JR, Hogan A, Paschalis V, et al. Mapping the interaction between eukaryotic initiation factor 4A (eIF4A) and the inhibitor hippuristanol using carbene footprinting and mass spectrometry. Proteomics. 2021;21(21-22). doi:10.1002/pmic.202000288