1. Outline Identification –2D gels (!) –MS (mass/seq) vs. MS/MS (mass/charge) Spectrum search –Modifications (later) –Resources: ExPASy –DBs: UniProt/SwissProt, Genpept, IPI Structure –Primary/secondary/tertiary –Crystallography vs. NMR –Disordered proteins (cool!) –Structure alignment + search: DALI, SALAMI, VAST –Prediction (CASP) Homology vs. threading vs. ab initio –DBs: PDB, SCOP, CATH, InterPro Domains + families –Family (large/2+ domain similarity), domain (local independent unit), motif (small target site) –Charge, accessibility, and conservation –DBs: Pfam, SMART, ProSite Localization –Direct assessment vs. prediction TM, secreted, localization signals –DBs: PSORT (insanely comprehensive), YPL 1

2. Protein localization Proteins may be localized to intracellular compartments, cytosol, the plasma membrane, or they may be secreted. Many proteins shuttle between multiple compartments. A variety of algorithms predict localization, but this is essentially a cell biological question. Page 240

3. Protein Localization: YPL 3

4. Protein Localization: PSort 4

5. Outline Identification –2D gels (!) –MS (mass/seq) vs. MS/MS (mass/charge) Spectrum search –Modifications (later) –Resources: ExPASy –DBs: UniProt/SwissProt, Genpept, IPI Structure –Primary/secondary/tertiary –Crystallography vs. NMR –Disordered proteins (cool!) –Structure alignment + search: DALI, SALAMI, VAST –Prediction (CASP) Homology vs. threading vs. ab initio –DBs: PDB, SCOP, CATH, InterPro Domains + families –Family (large/2+ domain similarity), domain (local independent unit), motif (small target site) –Charge, accessibility, and conservation –DBs: Pfam, SMART, ProSite Localization –Direct assessment vs. prediction TM, secreted, localization signals –DBs: PSORT (insanely comprehensive), YPL 5

6. Outline Stable interactions –Y–Yeast 2-hybrid –A–Affinity chromatography/MS (TAP-tagging) –O–Others: Co-IP, SPR, FRET –D–DBs: BioGRID, IntAct, DOMINE, and friends Transient interactions (modifications) –T–The big ones: phosphorylation, glycosylation –P–Peptide additions: ubiquitination, neddylation, sumoylation –O–Others: acylation, alkylation, etc. etc. etc. –D–DBs: PhosphoSite, dbPTM, PHOSIDA, Unimod, RESID… 6

7. Stable PPIs 7 Most proteins have friends

8. Detecting PPIs: Yeast 2-Hybrid 8 http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/Yeast2H.jpg http://genomebiology.com/2005/6/3/210/figure/F2 http://cmbi.bjmu.edu.cn/cmbidata/proteome/method/html/2hybrid02.files/Image16.jpg http://www.specmetcrime.com/yeast%20two%20hybrid%20approach%20.gif http://cmbi.bjmu.edu.cn/cmbidata/proteome/method/hybrid02.jpg http://en.wikipedia.org/wiki/ File:Two_hybrid_assay.svg

9. Detecting PPIs: Yeast 2-Hybrid 9 2000 2001 20% overlap ?

10. Detecting PPIs: Yeast 2-Hybrid So why aren’t we done yet? –False negatives Must localize to nucleus (membrane proteins = bad) Must express and more or less function in yeast –(Or other compatible expression/reporter vehicle) Fusion protein must maintain function Conditions must be right –False positives “Sticky” proteins (common!) Reporter autoactivation 10

11. Detecting PPIs: Yeast 2-Hybrid When yeast doesn’t mean. 11 2005 2003 2004

12. Detecting PPIs: Chromatography/MS Mass specs are good at identifying individual proteins –And mixtures of proteins –Why not identify mixtures of proteins that bind to individual proteins? 12 http://www.nature.com/nrm/journal/v3/n5/images/nrm804-f4.jpg

13. Detecting PPIs: Chromatography/MS 13 http://www.nature.com/nrm/journal/v4/n1/images/nrm1007-f1.jpg 2002

14. Detecting PPIs: Chromatography/MS 14 2002 2006

15. Detecting PPIs: Other technologies 15 http://www.cellmigration.org/resource/proteomics/images/flagpurifn.png http://www.piercenet.com/media/co-immunoprecipitation-700px.jpg Co-Immunoprecipitation http://www.nature.com/nrd/journal/v1/n7/images/nrd838-f2.gif Surface Plasmon Resonance http://zeiss-campus.magnet.fsu.edu/tutorials/spectralimaging/fretbiosensors/fretbiosensorsfigure1.jpg Fluorescence Resonance Energy Transfer

16. Downloading PPIs 16 dip.doe-mbi.ucla.edumips.helmholtz-muenchen.dewww.hprd.orgmint.bio.uniroma2.itwww.ebi.ac.uk/intactwww.thebiogrid.orgbond.unleashedinformatics.comdomine.utdallas.edu

17. Outline Stable interactions –Yeast 2-hybrid –Affinity chromatography/MS (TAP-tagging) –Others: Co-IP, SPR, FRET –DBs: BioGRID, IntAct, DOMINE, and friends Transient interactions (modifications) –The big ones: phosphorylation, glycosylation –Peptide additions: ubiquitination, neddylation, sumoylation –Others: acylation, alkylation, etc. etc. etc. –DBs: PhosphoSite, dbPTM, PHOSIDA, Unimod, RESID… Analysis of network… –…structure –…clustering (protein complex discovery) –…search –…alignment –…evolution –Resources: Cytoscape, Osprey, VisANT, Ondex, BiologicalNetworks, NeAT 17

18. Stable vs. Transient Interactions 18

19. Stable vs. Transient Interactions 19 Chen 2008

20. Transient interactions: phosphorylation 20 Mann 2003 http://www.websters-online- dictionary.org/images/wiki/wikipedia/commons/thumb/c/cd/Phosph orylated_serine.png/180px-Phosphorylated_serine.png http://bl1231.als.lbl.gov/2010/11/03/Hammel_fig3.jpg

21. Transient interactions: phosphorylation 21 2010 (!)

22. Transient interactions: glycosylation 22 Wildt 2005

23. Transient interactions: ubiquitination 23 http://en.wikipedia.org/wiki/Ubiquitin

24. Transient interactions: ubiquitination Does a known ubiquitinator bind a substrate? Does a substrate acquire ubiquitins? Does a substrate accumulate in a proteasome mutant? Does extra substrate hurt a proteasome mutant? 24 Liu 2010 2006 2009 2007 2009 2008 2007 2008 2009

25. Transient interactions: sumoylation and neddylation 25 SUMO: Small Ubiquitin-like ModifierNEDD8: Neural-precursor-cell-expressed developmentally down-regulated 8 http://www.nature.com/embor/journal/v9/n10/images/embor2008183-f1.jpg http://www.nature.com/nrm/journal/v8/n12/images/nrm2293-f2.jpg

26. Transient interaction DBs 26 www.phosphogrid.org phospho.elm.eu.org www.phosida.de www.phosphosite.org www.phosphopep.org dbptm.mbc.nctu.edu.tw www.unimod.org www.ebi.ac.uk/RESID www.cbs.dtu.dk/databases/OGLYCBASE ubiprot.org.ru scud.kaist.ac.kr sumosp.biocuckoo.org