Carbene Footprinting

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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.

Photene 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.

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)

What is Carbene Footprinting?

Carbene Footprinting is a powerful photochemical labelling technique used to map the binding sites of biological products with small molecules, peptides or proteins. It can be used to generate high-resolution binding site data, providing a critical insight into the mechanism of action for your products in development.

Carbene Footprinting has been used by our pharmaceutical, biotherapeutic and agrochemical industry partners to generate critical data for the following:

  • Protein-Protein Binding Site Analysis
  • Protein-Peptide Binding Site Analysis 
  • Protein-Ligand Binding Site Analysis
  • Membrane Protein Interaction Studies
  • Antibody-Antigen Epitope & Paratope Mapping
  • Molecular Glue Binding Site Analysis
How does it work?

We use a diazirine species, which upon irradiation with near-UV light generates a highly reactive carbene species. This carbene, covalently reacts with the accessible amino acids on the protein’s surface on a nanosecond timescale, generating a snapshot of the protein’s interactions.

When the protein forms a complex with its binding partner, the carbene species can no longer access the amino acids at the binding interface. Therefore, comparing the data for the protein in the presence and absence of its binding partner allows us to identify the binding site, as show below. 

After samples are labelled, they are then analysed via a bottom-up LC-MS/MS proteomic workflow. As the carbene species remains covalently bound to the protein after digestion, differences in mass of the corresponding peptides observed provide critical structural information. This information is then used to identify the binding site(s) of the sample.


“We have been extremely impressed with the collaboration, allowing us to access a powerful labelling technology for binding site determination. We have used this approach for studying multiple compound series with our target, which has been very challenging for traditional high-resolution structural approaches. The information generated has been extremely valuable for progressing our discovery programme”


Jonathon Hopper

Vice President Platforms & Founder, OMass Therapeutics

Technology Comparison

Relative to high-resolution techniques such as Nuclear Magnetic Resonance (NMR), X-Ray Crystallography and Cryogenic Electron Microscopy (Cryo-EM), mass spectrometry based techniques such as HDX, FPOP and Carbene Footprinting offer a lower spatial resolution.

However, ultra high-resolution data is often not necessary during the discovery and lead optimisation stages. Instead, amino acid level resolution offered by mass spectrometry based techniques offer a better balance of high resolution (amino acid level), speed, price and quantity of sample needed for the study. 

MS (Carbene Footprinting)
Sample Quantity
Amino Acid

There are several mass spectrometry based techniques used to map binding sites, including Hydrogen-Deuterium Exchange (HDX) and Flash Photooxidation of Proteins (FPOP). However, both of these techniques suffer from inherent limitations. Carbene Footprinting can overcome many of these limitations, as outlined in the Table below. 

Carbene Footprinting
Not Damaging*
Data Complexity

*Literature indicates the exposure of proteins to 248nm wavelength light and Hydrogen Peroxide are potentially damaging to protein structure. ± HDX requires the data analysis of for multiple time points whilst FPOP produces multiple different modification values, both adding to the data complexity. Carbene Footprinting generates a single modification and analyses a single time point.

HDX has two defining limitations. The first is the fact that the Hydrogen-Deuterium exchange itself is a reversible process, and therefore the sample needs to be quenched to avoid this back exchange. Therefore, harsh pH conditions and low temperatures are introduced to limit the effects of this. However, as a result digestion of the samples can only be performed using enzymes that tolerate these conditions, and is therefore primarily limited to pepsin. In addition, higher resolution data is difficult to achieve because of the lack of ability to perform MS/MS experiments due to H/D scrambling effects. 

FPOP on the other hand, requires exposing the sample to Hydrogen Peroxide, which evidence suggests can be damaging to the protein structure. Moreover, activation of the Hydrogen Peroxide to form the OH radical species uses 248 nm wavelength light, which is absorbed by aromatic residues and itself can impact the bound protein complex. 

Want to hear more?

Interested in hearing more about Carbene Footprinting and how it can benefit your research, discovery or lead optimisation programmes? Click here to get in touch with one of our team.