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Sequencing of Mammalian Genomes Predicts 30,000 genes

Sequencing of Mammalian Genomes Predicts 30,000 genes. 2001. 2002. What is the Proteome. All the proteins expressed in a particular cell or tissue. By definition this will vary by tissue and cell type. Cardiovascular Neuromuscular Islet cell Endothelial cell Muscle cell.

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Sequencing of Mammalian Genomes Predicts 30,000 genes

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  1. Sequencing of Mammalian Genomes Predicts 30,000 genes 2001 2002

  2. What is the Proteome • All the proteins expressed in a particular cell or tissue. • By definition this will vary by tissue and cell type. • Cardiovascular • Neuromuscular • Islet cell • Endothelial cell • Muscle cell

  3. Why Study Proteomics? • Gives a better understanding of the function of gene products. • Allow for the design of rational drug therapies. • Provide new and specific markers of disease.

  4. Will Proteomics Provide a Stronger Means to Stratify Disease YES Greater Complexity=Greater Specificity

  5. Protein Modifications Dictates Function and Intracellular Localization Phosphorylation Glycosolation Ubiquitination Cleavage Lipid etc.

  6. 30,000 Genes How Many Proteins? 30,000 genes 4-6 splice variants 120,000 possible mRNAS >200 Post-translational Modifications 24,000,000 Possible Protein Isoforms 2,000,000 Predicted Proteins

  7. How Gene and Protein Expression is Regulated or Modified from Transcription to Post-translation

  8. Synaptojanin 2

  9. BIOMARKER CHANGE +/- IDENTITY ? SPECIFIC EASE BODY FLUID PROTEIN INTERACTION PRESENCE ISOFORMS MODIFICATIONS FUNCTION Biomarker ID vrs Protein ID

  10. Using Proteomics to Identify Biomarkers of Disease

  11. How do You ID Biomarkers • 2D-PAGE  MALDI-TOF MS • ICAT  MALDI-TOF MS • SELDI-TOF MS

  12. Proteomic flow 1D and 2D gels Proexpress Imager Propicker Progest ProMS Q Star XL -MS/MS-TOF Biacore Vision Station

  13. SELDI-TOF MS Real Time PCR Centre for Proteomic and Genomic Studies vision workstation agilent lab-on-chip Q-Star-XL MS/MA-TOF Protein Isoform Characterization Lab

  14. ICAT(Isotope Coded Affinity Tag) Deuterium Thiol Reactive Group Biotin Linker

  15. ICAT Provides Relative Levels of Expression Between Two Proteins Which is Reflected by the Ratio of the Amount of Peptide

  16. Surface Enhanced Laser Desorption Ionization Time of Flight (SELDI-TOF) Protein Chip

  17. Protein Chip

  18. Different Protein Profiles from Different Chip Surfaces

  19. Laser Capture Microscopy Allows for the Dissection of Cells Directly from Sectioned Tissues

  20. LCM of Normal and Cancerous Prostate

  21. Protein Profile from Prostrate Cancer Tissue Procured by LCM

  22. Do we Need all Three Approaches? • 2D-PAGE  MALDI-TOF MS • Greatest resolving power • Large data base • Labor and time consuming • ICAT  MALDI-TOF MS • Mostly abundant proteins • Needs cysteine residue • SELDI-TOF MS • Resolves from 8-50 kDa • Small amounts of sample

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