Protein CrossLinking and Computational Techniques for the Identification of Protein Complexes

1
Protein Cross-Linking and Computational
Techniques for the Identification of Protein
Complexes

• 1Jana A. Lewis, 2Gregory B. Hurst, and 2,3James
  L. Stephenson, Jr.
• 1Department of Chemistry, Lawrence University
• ERULF/GLCAProgram
• 2Chemical Sciences Division, Oak Ridge National
  Laboratory
• 3Research Triangle Institute

2
Protein Structure Determination Methods

• X-ray Crystallography
• Nuclear Magnetic
• Resonance Spectroscopy

• Theoretical Calculations
• Mass Spectrometry

As of 12/11/01 posted at http//www.rcsb.org/pdb/h
oldings.html
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Objectives

• Protein complex identification using protein
  cross-linking and computational techniques
• Find inter/intramolecular distance estimates
• Use distance constraints in computational
• structure calculations
• Use a known system to develop a robust method

4
Why Use Hemoglobin as a Model?

• Well-characterized protein
• complex
• High resolution NMR
• structure
• X-ray crystal structure
• Known inter/intra-
• molecular distances
• Known amino acid
• sequence
• Four subunits

Crystal structure of bovine hemoglobin
1FSX (http//www.rcsb.org/pdb/)
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Protein Cross-Linking

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Cross-Linker Characteristics

• Chemical specificity
• Length of spacer arm
• Homobifunctional/
• heterobifunctional
• Functional groups for purification

• Solubility
• Membrane permeability
• Chemical or photochemical reaction

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Methods Behind Our Madness
Tetramer
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MALDI Mass Spectrometry
MALDI on Intact Product
MALDI on Tryptic Digest
5.59 BS3Hb, pH 7.4
Counts
Counts
a-chain
22.4 BS3Hb, pH 8.2
Excessive cross-linking
m/z
m/z
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Peak Identification by FTICR
MALDI on Digest
FTICR
MH
Observed m/z 3327.731 FTICR Calculated
m/z 3327.785 BS3 8-29b 8-16a
2.24 BS3Hb
Intensity
3327.738 116-145b
Da
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The Quest Continues
MS on Tryptic Digest
Possible cross-linked peptide for MSn
Intensity
m/z
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Conclusions

• Lower protein and cross-linker concentrations
  lead to fewer incorrect distance constraints.
• The optimum pH is around 7 for the hemoglobin and
  BS3 cross-linking reactions.
• Use FPLC to isolate the cross-linking reaction
  products before the trypsin digestion.
• FTICR-MS provides high accuracy masses to
  identify tryptic peptides versus cross-linked
  products.

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Future Plans

• Cross-link peptides with known sequences
• Chromatographic separation of peptides before
• mass spectrometry
• Use multiple stage mass spectrometry experiments
  
• and computational tools to check product
  identities
• Use robotics to speed the screening of
  cross-linking
• reaction products.

13
Acknowledgments

• Department of Energy
• ORNL Seed Money
• Jonathan Bundy
• Nathan Verberkmoes
• Keiji Asano
• Robert Hettich

• GLCA/ACM Oak Ridge Science Semester Program
• ERULF Program
• Wayne Wolsey
• Wendy Williams
• Linda Holmes
• Cheryl Terry
• Kathy Ketner