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![[Figure 1]](ig5064fig1mag.jpg)
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Figure 1
Schematic diagram of protein X-ray footprinting experiments. Control (e.g. apo) and experimental (e.g. ligand-bound) samples are exposed to an X-ray beam for microsecond to millisecond timescales. Exposed samples are digested with proteases to generate peptides, which are separated via ultra-performance liquid chromatography (UPLC). Electrospray ionization (ESI) is used to ionize the peptides, which enter the mass spectrometer where they are separated according to their mass-to-charge (m/z) ratios (MS) and are subsequently fragmented through collision with gas molecules (MS/MS). The fragmentation pattern is unique to each peptide, so MS/MS scans can be used to identify unmodified and modified peptides/residues within the sample. Extracted ion chromatograms (EICs) are used to quantify the amount of unmodified and modified peptides observed. The fraction of unmodified peptide remaining is calculated at each exposure time for each modified peptide/residue and plotted to produce dose-response curves, which are fit to a single-exponential function to determine first-order rate constants. The calculated rate constants in the control (kapo) and experimental (kligand) samples are compared in order to determine regions of the protein that are important for function. |
![[Figure 1]](ig5064fig1.jpg)
 | JOURNAL OF SYNCHROTRON RADIATION |
ISSN: 1600-5775
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