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Top Down Proteomics: A Datasource for PRO

Top Down Proteomics: A Datasource for PRO. Paul Thomas, Northwestern University Ryan Fellers, Cecilia Arighi ( UDel /PIR/ UniProt ) The Consortium for Top Down Proteomics 16 May 2013. Protein Variability in Higher Proteomes: The Age of Protein Isoforms. Sources of variability

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Top Down Proteomics: A Datasource for PRO

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  1. Top Down Proteomics:A Datasource for PRO Paul Thomas, Northwestern University Ryan Fellers, Cecilia Arighi (UDel/PIR/UniProt) The Consortium for Top Down Proteomics 16 May 2013

  2. Protein Variability in Higher Proteomes:The Age of Protein Isoforms • Sources of variability • Alternative Splices • Endogenous Cleavages • Alternative Promoter Usage Base Isoform Insertion or Alternative Sequence Isoform: Large changes to amino acid sequence

  3. Protein Variability in Higher Proteomes:The Age of Protein Isoforms and Proteoforms Proteoform: The complete set of modifications on an amino acid sequence http://dx.doi.org/10.1038/nmeth.2369

  4. What’s in a name? You can’t please everyone!

  5. Technical Hypothesis Measurement of intact proteins (isoforms and proteoforms) will provide strong correlations between mass spectrometric data and complex phenotypes.

  6. Differences: The measurement challenge C N N C

  7. Top Down vs. Bottom Up Approaches to DNA-Predicted Protein Sequence Analysis

  8. 3. “Back end” data processing and informatics DEVELOPMENT OF A GENERAL MEASUREMENT PLATFORM FOR INTACT, MODIFIED PROTEINS 1. “Front End” Separations 2. Automated Instrumentation

  9. Top Down Proteomics Work Flow _ 200 kDa + – + GELFREE – MW based separation (Optional) isoelectric focusing 10 kDa Cell lysate or nuclei Online nano-LC-MS/MS: 12 T/Orbitrap LTQ-FTMS Sample cleanup (SDS removal)

  10. Top Down Proteomics and Plants

  11. A. thalianaThylakoid Lumen PO:0020039

  12. Similar Proteins

  13. www.topdownproteomics.org

  14. Proteoform Repository

  15. PFR2217

  16. Senescence

  17. Chemotherapy-Induced Accelerated Senescence Control CHEBI:27656 Treatment: 25nM camptothecin for 24 hours Senescent Early Escape? Roberson, R. S et al. Cancer research2005, 65, 2795-803. Wang, Q. Wu, P. C. et al. International journal of cancer. 2011, 128, 1546-58.

  18. HMGA1 Isoforms Control P17096-1 HMGA1a: Senescent P17096-2 HMGA1b: Escape Acetylation Phosphorylation Methylation

  19. PRIDE, Modified HMGA1

  20. Glyco-Proteoforms OH ApoC-III ApoC-III ApoC-III Unmodified MOD:????? MOD:????? Linear Branched

  21. Proteomics Center of Excellence Northwestern University http://pce.northwestern.edu

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