Structural proteomic of macromolecular assemblies using oxidative footprinting and mass spectrometry

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Structural proteomic of macromolecular assemblies
using oxidative footprinting and mass
spectrometry

• Jing-Qu Guan and Mark R. Chance
• Case Western Reserve University

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Outline

• General strategy
• -gtClassical footprinting approaches
• -gtMS approaches to structure
  determination
• Examples
• Concluding remarks

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Classical footprinting approaches
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Classical footprinting approaches

• chemical/enzymatic/modification
• back bone/bases/side chains

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MS approaches to structure determination

• Gentle ionization methods
• e.g. ESI / MALDI
• Highly sensitive detection methods
• femtomole
• Hydroxyl radical (reaction mainly on side-chains)

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Detecting and Analysis

• HPLC-MS
• fragments and modified/unmodified
• Dose-response curve
• unmodified fraction vs. exposure time
• Oxidation sites are detected by MS/MS

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Dose-response curve
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Oxidation sites detection

• CID -gtb-type y-type fragment ions
• Unmodified vs. modified peptide MS spectra

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Application

• Protein footprinting coupled with mass
  spectrometry is an powerful technique when
  coupled with existing structural data, to
  understand ligand-induced conformational models
  of structure

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the binding interface of transferrin C-lobe on
its receptor (TF TFR)

• Crystallographic structure available
• Nature of domain-domain interaction that drive
  complex formation is still controversial

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Computational model of the Sml1 protein

• NMR is applied
• two large a-helices that are oriented in an
  anti-parallel manner ,but no tertiary structure
  well-defined.
• Footprinting --- de novo method

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Computational model of the Sml1 protein

• denatured Sml1 was used as a control molecule
• (At least 18 oxidation sites were identified
  )
• then compared with reactivity data for the native
  protein
• (Pro22, Phe33, Phe87 and Phe106 were not
  oxidized in the native protein )
• residues determined to be either accessible or
  buried were analyzed for consistency with models
  of the protein sequence low energy model

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conclusion

• sensitive probe of solvent accessibility
• femtomoles to picomoles of material
• monitor multiple sites within multi-protein
  complexes simultaneously
• No intrinsic limitations with respect
• to the size of the macromolecules

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• Discussion