Proteomics Technologies with a Focus on Mass Spectrometry

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Proteomics Technologies with a Focus on Mass
Spectrometry

• John Carrithers

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Presentation Goals

• Increase understanding of the Proteome
• Share some of the proteomic technologies at work
• Provide basis theory of mass spectrometry in the
  field of proteomics
• Show current applications of the technology

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The Central Dogma of Molecular Biology
DNA mRNA Proteins
Genomics
Now What?
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The Central Dogma of Molecular Biology
DNA mRNA Proteins
Genomics
Genomic Expression
Proteomic Expression
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Why is Proteomics the Logical Next Step?
Or More?
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Technical Approaches

• Separation of Complex biomolecular mixtures
  electrophoresis and chromatography
• Structural Analysis mass spectrometry
• Informatics proteome databases

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Slides courtesy of William M. Pierce,
Jr. Professor of Pharmacology and
Toxicology Professor of Chemistry Director -
Biomolecular Mass Spectrometry Core Laboratory
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Preparation

• Liquid Liquid Extraction
• Formation of Derivatives
• Affinity enrichment
• To obtain sample for analysis

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Separation

• HPLC
• Gas Chromatography
• Electrophoresis
• Capillary
• 1D and 2D SDS-Page Electrophoresis
• Allows for the separation of proteins
• Individual Proteins available for analysis

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Typical 2D gel electrophoresis
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Ionization

• Ionization is the key to mass spectrometry of
  large biomolecules
• Hard ionization techniques would damage
  biomolecules
• Soft Ionization (ES and MALDI) provide a pathway
  to Mass Spec historically limited to small
  molecules, easy to ionize

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Soft Ionization Mass Spec

• ESI and MALDI
• Very important for large biomolecules
• Allows for structural analysis qualitative
• fingerprinting of molecules

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Basic Theory of Mass Spectrometry
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Mass Spec Used in Proteomics
http//www.nature.com/nature/journal/v422/n6928/fi
g_tab/nature01511_F2.html
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Typical Proteomics Experiment
http//www.nature.com/nature/journal/v422/n6928/fi
g_tab/nature01511_F2.html
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Other Techniques

• Proteomics technology is not limited to mass spec
• X-ray crystallography
• Microarrays Biochip arrays
• Allow for many reactions to take place in a small
  space
• Informatics protein databases

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The SELDI Process and BioChip Arrays
1) Expose BioChip to sample
containing the target biomarker proteins
2) Develop BioChip (retains captured target
proteins and washes away others )
3) Read BioChip by SELDI process
(Surface-Enhanced Laser Desorption/Ionization)
Protein Fingerprint Database
Patient Sample
Molecular Weight
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SELDI Protein BioChip
---for multiple biomarkers
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Application of Technology

• Sequencing
• ESI and tandem MS (MS-MS)
• Mass Measurement
• Extreme precision

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Precision of Mass Measurement
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References

• William M. Pierce, Jr. - Director - Biomolecular
  Mass Spectrometry Core Laboratory
• Nature Proteomics www.nature.com/nature/proteomics
  
• Cambridge University Mass Spectrometry Server
  Theory Section http//www-methods.ch.cam.ac.uk/met
  h/ms/theory/index.html
• British Mass Spectrometry Society
  http//www.bmss.org.uk/what_is/whatisframeset.html
  
• Nature. Mass spectrometry-based proteomics R
  Aebersold M Mann
• Nature. From genomics to proteomics M Tyers M
  Mann