Protein CrossLinking and Computational Techniques for the Identification of Protein Complexes - PowerPoint PPT Presentation

Loading...

PPT – Protein CrossLinking and Computational Techniques for the Identification of Protein Complexes PowerPoint presentation | free to view - id: 20693e-YmQzN



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

Protein CrossLinking and Computational Techniques for the Identification of Protein Complexes

Description:

Protein Cross-Linking and Computational Techniques for the ... Wendy Williams. Linda Holmes. Cheryl Terry. Kathy Ketner. Department of Energy. ORNL Seed Money ... – PowerPoint PPT presentation

Number of Views:354
Avg rating:5.0/5.0
Slides: 14
Provided by: A1265
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: 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
3
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/)
5
Protein Cross-Linking

6
Cross-Linker Characteristics
  • Chemical specificity
  • Length of spacer arm
  • Homobifunctional/
  • heterobifunctional
  • Functional groups for purification
  • Solubility
  • Membrane permeability
  • Chemical or photochemical reaction

7
Methods Behind Our Madness
Tetramer
8
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
9
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
10
The Quest Continues
MS on Tryptic Digest
Possible cross-linked peptide for MSn
Intensity
m/z
11
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.

12
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
About PowerShow.com