1. Thanks to: George Church (Harvard GTL & CEGS Centers) 5-Jan-2006 HPCGG Landsdowne 2 PM Personal Genomics meets Quantitative Proteomics NHGRI Seq Tech 2004: Agencourt, 454, Microchip, 2005: Nanofluidics, Network, VisiGen Affymetrix, Helicos, Solexa-Lynx

2. "Open-source" Personal Genome Project (PGP) Harvard Medical School IRB Human Subjects protocol submitted 16-Sep-2004, approved Aug 31, 2005. Gradual plan. Start with "highly-informed" individuals consenting to non-anonymous genomes & extensive phenotypes (medical records, imaging, omics). Cell lines in Coriell NIGMS Repository Diploid genome subsets at $0.1/kb, <3E-7 FP Errors How? Polony bead Sequencing-by-Ligation (SbL)

3. Analyses of single chromosomes (single cells, RNAs, particles) (1) When we only have one cell as in Preimplantation Genetic Diagnosis (PGD) or environmental samples (2) Candidate chromosome region sequencing (3) Prioritizing or pooling (rare) species based on an initial DNA screen. (4) Multiple chromosomes in a cell or virus (5) RNA splicing (6) Cell-cell interactions (predator-prey, symbionts, commensals, parasites)

4. CD44 Exon Combinatorics (Zhu & Shendure) Alternatively Spliced Cell Adhesion Molecule Specific variable exons are up-or-down-regulated in various cancers (>2000 papers) v6 & v7 enable direct binding to chondroitin sulfate, heparin… Zhu,J, et al. Science. 301:836-8.

5. Zhu J, Shendure J, Mitra RD, Church GM. Science 301:836-8. Single molecule profiling of alternative pre-mRNA splicing. Eph4 = murine mammary epithelial cell line Eph4bDD = stable transfection of Eph4 with MEK-1 (tumorigenic) CD44 RNA isoforms

6. Molecular Weight Assessment of Proteins in Total Proteome Profiles Using 1D-PAGE and LC/MS/MS. Proteome Sci. 3:6 (2005) Ahmad R, Nguyen DH, Wingerd MA, Church GM, Steffen MA. Candidates for alternative splicing (AS), endoproteolytic processing (EPP), & post-translational modifications (PTMs) in Lymphoblastoid cells Protein Name Predicted MW Observed MW Difference before & after leader cleavage Cytochrome c oxidase subunit IV isoform 1 19577 2582 205 NADH dehydrogenase 21750 5084 334 Coproporphyrinogen oxidase 50175 13632 357 MHC II, DQ  1 29733 25896 404 NADH (ubiquinone) Fe-S protein 2 52545 48185 815 Mito short-chain enoyl-coA hydratase 1 31371 27499 901 Peptidylprolyl isomerase B (cyclophilin) 23742 19360 940

7.  -Glc-1P ADP-Glc  -1,4-glucosyl-glucan glycogen Central Carbon Metabol. glgC glgX glgA glgB glgP Zinser et al. unpubl. Light regulated Circadian metabolism

8. Viral Photosynthetic Proteins Podovirus P-SSP7 46 kb PCHLIPsFdD1 12kb 24kb PCHLIPsFdD1 12kb 24kb ~500bp HLIPsD1D2 6.4kb2.8kb ~500bp Myovirus P-SSM4 181 kb HLIPsD1D2 6.4kb2.8kb Lindell, Sullivan, Chisholm et al. 2004 HLIPD1 Myovirus P-SSM2 255 kb

9. Photosynthesis genes in marine viruses yield proteins during host infection. Nature 2005 438:86-9. Lindell D, Jaffe JD, Johnson ZI, Church GM, Chisholm SW.

10. Photosynthesis genes in marine viruses yield proteins during host infection. Nature 2005 438:86-9. Lindell D, Jaffe JD, Johnson ZI, Church GM, Chisholm SW. 15N 13C synthetic standards host phage

11. Improving MS Peptide Coverage ? Ionization efficiency X Ions outside the mass range of the analyzer ? Chromatographic behavior ? Sample preparation bias X Instrument duty cycle Improve Spectra interpretation over current algorithms –Details of fragmentation patterns –Dipeptide P, DE/KR, V.G intensity effects –B & Y ions unequal & co-dependent –More intense ions in middle of peptides MDQuest: Mike Chou, Dan Schwartz, Steve Gygi, Josh Elias http://gygi.med.harvard.edu/dpsp/

12. SEQUEST vs MDQUEST Performance

13. MapQuant is a program designed to isolate unique organic species and quantify their relative abundances from an LC/MS experiment. Scheme: Data from an LC/MS experiment are analyzed after being formatted into a data structure called a 2-D map, analogous to a gray-scale image. Scan number: N N+1 N+2 N+3 2-D peptide map time or scans m/z units

14. 2-D map Retention time m/z units MapQuant Gives a List of All Organic Species In the Sample MapQuant

15. MapQuant is a program designed to isolate unique organic species and quantify their relative abundances from an LC/MS experiment. Scheme: Data from an LC/MS experiment are analyzed after being formatted into a data structure called a 2-D map, analogous to a gray-scale image. Scan number: N N+1 N+2 N+3 2-D peptide map time or scans m/z units

16. 2-D map Retention time m/z units MapQuant Gives a List of All Organic Species In the Sample MapQuant

17. Leptos et al. Proteomics 2006

18. MapQuant is publicly available at http://arep.med.harvard.edu/mapquant.html

19. Leptos et al. Proteomics 2006

21. retention time (in min) m/z units EKLAVSAR QEPERSEK DAFLSGER ? ? ? MapQuant gives me a list of all organic species in the sample BUT WHAT ARE THEIR IDENTITIES?

22. MapQuant identifies approx. 2x10 4 organic species per LC/MS experiment. ONLY ~ 500 (3%) organic species have fragmentation (CID) spectra and hence sequence IDs retention time (in min) EKLAVSAR QEPERSEK DAFLSGER ? ? ? m/z units Dealing With Many Peptides (Organic Species) 22 = CID spectrum or MS/MS event

23. Dealing With Many Peptides (Organic Species) retention time (in min) EKLAVSAR QEPERSEK DAFLSGER ? ? ? Database of 11845 peptides from ALL LC/MS experiments carried out on Prochlorococcus samples (rt, m/z) coordinates m/z units

24. TOTAL NUMBER OF ORFS: 1742 1314 539 522792 17 Protein Distribution Among Experiments

25. Sequence Coverage of the Protein groES

26. Summary Proteome Sci. 3:6 (2005) Ahmad R, Nguyen DH, Wingerd MA, Church GM, Steffen MA. Open Personal Genome Project (PGP) including Proteomics Single molecule RNAs for alternative splicing (AS) Gel –MS methods for endoproteolytic processing MapQuest for MS quantitation without isotopic labeling http://arep.med.harvard.edu

27. Thanks to: George Church (Harvard GTL & CEGS Centers) 5-Jan-2006 HPCGG Landsdowne 2 PM Personal Genomics meets Quantitative Proteomics NHGRI Seq Tech 2004: Agencourt, 454, Microchip, 2005: Nanofluidics, Network, VisiGen Affymetrix, Helicos, Solexa-Lynx