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Proteomics Technologies with a Focus on Mass Spectrometry

Proteomics Technologies with a Focus on Mass Spectrometry. John Carrithers. 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

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Proteomics Technologies with a Focus on Mass Spectrometry

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  1. Proteomics Technologies with a Focus on Mass Spectrometry John Carrithers

  2. 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

  3. The Central Dogma of Molecular Biology DNA mRNA Proteins Genomics Now What?

  4. The Central Dogma of Molecular Biology DNA mRNA Proteins Genomics Genomic Expression Proteomic Expression

  5. Why is Proteomics the Logical Next Step? Or More?

  6. Technical Approaches • Separation of Complex biomolecular mixtures – electrophoresis and chromatography • Structural Analysis – mass spectrometry • Informatics – proteome databases

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

  8. Preparation • Liquid – Liquid Extraction • Formation of Derivatives • Affinity enrichment • To obtain sample for analysis

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

  10. Typical 2D gel electrophoresis

  11. 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

  12. Soft Ionization Mass Spec • ESI and MALDI • Very important for large biomolecules • Allows for structural analysis – qualitative • “fingerprinting” of molecules

  13. Basic Theory of Mass Spectrometry +COH +CH3 CH3COCH3 CH3+COCH3 CH3C+OCH3 +COCH3 Ionization & Adsorption of Excess Energy Fragmentation (Dissociation) Detection Mass Analysis Sample Inlet

  14. Mass Spec Used in Proteomics http://www.nature.com/nature/journal/v422/n6928/fig_tab/nature01511_F2.html

  15. Typical Proteomics Experiment http://www.nature.com/nature/journal/v422/n6928/fig_tab/nature01511_F2.html

  16. 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

  17. 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 3 1 SELDI Protein BioChip ---for multiple biomarkers 2

  18. Application of Technology • Sequencing • ESI and tandem MS (MS-MS) • Mass Measurement • Extreme precision

  19. Precision of Mass Measurement

  20. 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/meth/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

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