1. Intro Spring 2009 Bioinformatiatics Proteomics

2. workflow Spring 2009 Bioinformatiatics Proteomics Workflow Sample Prep Sequencing Database Search Protein ID Protein Interactions

3. Identification Quantification General workflow of proteomics analysis External data sources taxonomy, ontologies, bibliography… Applications Systems biology (pathways, interactions..) biomarker- discovery, drug targets Proteins/peptides 2D gel image aquisition and storage MALDI, MS/MS Store peak lists and all meta data Digestion and/or separation PMF MS/MS DIGE LC-MS & Tags

4. Sequence data bases: EMBL Nucleotide Sequence Database GenBank UniProtKB/Swiss-Prot & TrEMBL Ensemble EST database PIR Identification Quantification General workflow of proteomics analysis Proteins/pe ptides Digestion and/or separation MALDI, MS/MS 2D Page data bases Swiss 2D PAGE, Gelbank, Cornelia, WordPAGE Make 2D Imaging tools : Melanie, PDQuest Progenesis Delta 2D Storing/ organising: Proteincsape MSight KEGG PDB DIP OMIM Reactome PROSIT Pfam SPIN BOND STRING AmiGO David PubMed MEDLINE Mascot Sequest Aldente Popitam Phenyx FindMod Profound PepFrag MS-Fit OMSSA Search XLinks TagIdent

5. General workflow of proteomics analysis Proteins/pe ptides Digestion and/or separation 2D Page data bases Make 2D Imaging Softwares: The ability to compare two gels (images) and then identify differently expressed spots Melanie PDQuest Progenesis Delta 2D Proteinscape –platform for storing, organizing data MSight -representation of mass spectra along with data from the separation 2D gel databases: Data integration on the web Image data and textual information Swiss 2D PAGE Gelbank Cornelia WordPAGE

6. Laser capture Spring 2009 Bioinformatiatics Laser-Capture Micro dissection, LMC Technique for selectively sampling certain cells within a tissue Biopsy Transfer film Glass slide Genomic/proteomic analysis Tissue sample Laser beam activates film Selected cells are transferred Tumor Cells Modified from “National Cancer Institute”, US National Institutes of Health: http://www.cancer.gov/cancertopics/understandingcancer/moleculardiagnostics/Slide29

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8. Fractionation Spring 2009 Bioinformatiatics Affinity Purification

9. 2D gels at Swissprot Spring 2009 Bioinformatiatics Swissprot ExPaSy Database 2D Electrophoresis

10. Template Spring 2009 Bioinformatiatics Protein Digestion Primary sequence must be accessible Denature – urea in solution or SDS in gel Reduce & alkylate disulfide bonds between cysteines dithiothreitol (DTT) & Iodoacetamide (IAA) Digest with enymes Purify peptide fragments

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12. codon Usage Spring 2009 Bioinformatiatics Standard Genetic Code (transl_table=1) AAs = FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG Starts = ---M---------------M---------------M---------------------------- Base1 = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG Base2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG Base3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG AAs = FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG Starts = ---M---------------M------------MMMM---------------M------------ Base1 = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGG G Base2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGG G Base3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCA G The Bacterial and Plant Plastid Code (transl_table=11) AAs = FFLLSSSSYYQQCC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG Starts = -----------------------------------M---------------------------- Base1 = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG Base2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG Base3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG The CiliateHexamita Nuclear Code (transl_table=6)

13. Unusual amino acids Spring 2009 Bioinformatiatics Unusual Amino Acids

14. phosphorylation Spring 2009 Bioinformatiatics Phosphorylation - signal transduction mRNA

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21. MS analysis

22. Antibody arrays Good for low-abundance proteins Problem is antibody specificity

23. Array-based protein interaction detection

24. Protein microarrays

25. Yeast Two-Hybrid System

26. How to organize information? Gene Ontology –Biological process Frequently from biochemical analyses In silico analysis –Molecular function Biochemical analysis –Cellular component Biochemical analysis GFP or other tagging

27. Interaction maps - Grid

28. challenges Complexity – some proteins have >1000 variants Need for a general technology for targeted manipulation of gene expression Limited throughput of todays proteomic platforms Lack of general technique for absolute quantitation of proteins

29. Protein Profiling 2D gel electrophoresis Difference gel electrophoresis (DIGE) LC-MS/MS using coded affinity tagging (ICAT, iTrac, SILAC..) ProteinChip Array (SELDI analysis) Antibody arrays Measure the expression of a set of proteins in two samples and compare them - Comparative proteomics

30. Intro Spring 2007 Bioinformatiatics RNA and Protein Structure Prediction

32. SSU Secondary Structure

33. Ribosome

35. -Beaudry & Joyce, Science, 1992 Frq. Of mutation (%; n=25) after 9 generations.

37. M13 vector sequence TATAGGGCGAATTGAATTTAGCGG or ATTAACCCTCACTAAAGGGACTAG to CCCTT

38. Pseudoknots

39. Hammerhead Ribozyme

40. tRNA Secondary Structure

41. RNA Tertiary Structure

45. Pyruvate Kinase

46. Human DNA clamp PCNA

47. Chou-Fasman Parameters