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![[Figure 4]](ms5002fig4mag.jpg)
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Figure 4
Schematic representation of protein footprinting using synchrotron radiolysis and mass spectrometry. The examples emphasize the protection formed in the interface of a protein–ligand complex as well as allosteric conformation changes that can result in increases in reactivity upon ligand binding; however, the comparison could be for any two (or multiple) functional states of the protein of interest. Two sets of samples, one free protein and the other a protein–ligand complex, are exposed to X-rays for different time intervals. The exposed samples are digested with specific digestion enzymes. The digested fragments are analyzed by ESI-MS to quantitate the extent of modification products and determine the fraction `unmodified' for a specific exposure time. A plot of fraction unmodified versus exposure time, known as the dose response plot, fit to a first-order function providing the rate of modification for the specific peptide. Comparisons of the dose response of the same peptide under different conditions provide structural information about ligand binding. MS/MS is used to determine the specific modification site within the peptide and provide side-chain-specific structural resolution. Synchrotron protein footprinting data are often used as one of the constraints in model building for complexes. |
![[Figure 4]](ms5002fig4.jpg)
 | JOURNAL OF SYNCHROTRON RADIATION |
ISSN: 1600-5775
