Application Note 56

Biological Advances Using the Protein Footprinting Method Covalent Protein Painting

John R. Yates III¹, Ahrum Son², T. Casimir Bamberger¹, Sandra Pankow¹

1. Department of Integrated Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037
2. Graduate School of Medical Science, University of Ulsan, Ulsan, Republic of Korea, 44610

Research techniques are emerging that focus on determining the confirmation of proteins within cells and tissues. One of these techniques, protein footprinting, uses covalent labeling to mark surface-accessible amino acids on proteins, which allows investigations into conformational changes of a protein, such as locating drug binding or epitope mapping. Like hydrogen-deuterium exchange (HDX), which replaces exchangeable hydrogens on proteins with deuterium, protein footprinting attaches a covalent tag to surface-accessible amino acids. In contrast to HDX, covalent labeling is permanent, which allows sample processing without the need to worry about D/H back-exchange. A potential drawback of protein footprinting has been that fewer labeling sites are available in a protein. To overcome this limitation, highly reactive reagents for covalent labeling (such as free radicals) have been employed to achieve more comprehensive coverage of protein surfaces. But the extreme reactivity of radicals, like oxygen, necessitates their generation in situ and leads to promiscuous labeling that makes data analysis challenging.

We have developed a very focused and labeling-efficient protein-footprinting method that utilizes the well-established proteomic chemistry of dimethyl tagging and provides quantitative data. This approach begins with the formation of a Schiff base at 
the ε-amine of lysine residues using formaldehyde, which is subsequently reduced with cyanoborohydride to create a stable dimethyl group. A key advantage of formaldehyde is its proven use in tissue preservation due to ability to rapidly diffuse into cells and tissues. Additionally, both formaldehyde and sodium cyanoborohydride can be synthesized with stable isotope labels, enabling isotope-defined tags with distinct masses.

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