XFMS - About

Overview

Chemical footprinting has been used for over five decades now as a method for obtaining structural information on biomolecules. The idea of using X-rays for footprinting, however, is relatively new. X-ray footprinting was first conceived and tried at the National Synchrotron Light Source at Brookhaven National Laboratory. Since then, the technique has grown in popularity and has been used to study structure and dynamics in a wide range of protein and nucleic acid systems, revealing information on ligand binding, protein-protein interactions, protein-nucleic acid interactions, and even on systems as complex as in vivo ribosome assembly. In the last decade, we have introduced this method at the Advanced Light Source synchrotron at Berkeley Lab, making it the second synchrotron X-ray footprinting facility in the United States.

Please note that starting in the Fall of 2027 when the ALS upgrade begins, we will be conducting X-ray irradiation at the Stanford Synchrotron Radiation Lab (SSRL) through a collaborative agreement. Proposals can be sent through the Molecular Foundry user portal as usual; beamline staff will irradiate samples for users at SSRL and will collect LCMS data for users at the ALS.

How it works

When X-rays impinge on a water-based buffer, hydroxyl radicals are created. These radicals will modify protein residues only where they are accessible to water. Therefore, if you can locate the modifications, you can determine a solvent accessible map of the protein. This is useful, for instance, for watching protein folding, or for determining protein interaction points. In practice, after exposure to X-rays, the protein samples are digested with proteases and then analyzed using LCMS to determine locations of modifications. If you are familiar with hydrogen-deuterium exchange (HDX) then you will have noted the similarity of footprinting to HDX. With HDX, the protein backbone is mapped, whereas here, the sidechains are mapped. In addition, with footprinting, the modifications are covalent, and so not limited by pH or temperature.

Why use a synchrotron

Other radiation sources (and chemical sources) can be used for footprinting. But synchrotrons provide a very intense and focused X-ray beam. This, it turns out, is very important for the footprinting experiment. An intense brief burst of radiation produces a high enough radical concentration in such a short amount of time such that modifications occur before other secondary damage affects the proteins. In addition, the brighter the beam, the less sample that is necessary. For our footprinting experiments, we use typically 5-10 micromolar concentrations of proteins, and about 200 microliters per sample.

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