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 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.

7. Differences: The measurement challenge N C N C

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

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

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

11. Top Down Proteomics and Plants

12. A. thaliana Thylakoid Lumen PO:0020039

13. Similar Proteins

14. www.topdownproteomics.org

15. Proteoform Repository

16. PFR2217

18. Senescence

19. Chemotherapy-Induced Accelerated Senescence Roberson, R. S et al. Cancer research 2005, 65, 2795-803. Wang, Q. Wu, P. C. et al. International journal of cancer. 2011, 128, 1546-58. Treatment: 25nM camptothecin for 24 hours Control Senescent Early Escape? CHEBI:27656

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

21. PRIDE, Modified HMGA1

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

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