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Intro

Intro. Bioinformatiatics. Spring 2009. Proteomics. workflow. Bioinformatiatics. Spring 2009. Proteomics Workflow. Sample Prep Sequencing Database Search Protein ID Protein Interactions. General workflow of proteomics analysis. Proteins/peptides. Digestion and/or separation.

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Intro

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  1. Intro Bioinformatiatics Spring 2009 Proteomics

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

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

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

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

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

  7. Template Bioinformatiatics Spring 2009

  8. Fractionation Bioinformatiatics Spring 2009 Affinity Purification

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

  10. Template Bioinformatiatics Spring 2009 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

  11. Template Bioinformatiatics Spring 2009

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

  13. Unusual amino acids Bioinformatiatics Spring 2009 Unusual Amino Acids

  14. phosphorylation Bioinformatiatics Spring 2009 Phosphorylation - signal transduction mRNA mRNA

  15. Template Bioinformatiatics Spring 2009

  16. Template Bioinformatiatics Spring 2009

  17. Template Bioinformatiatics Spring 2009

  18. Template Bioinformatiatics Spring 2009

  19. Template Bioinformatiatics Spring 2009

  20. Template Bioinformatiatics Spring 2009

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

  30. Intro Bioinformatiatics Spring 2007 RNA and Protein Structure Prediction

  31. SSU Secondary Structure

  32. Ribosome

  33. Ribosome

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

  35. M13 vector sequence TATAGGGCGAATTGAATTTAGCGG or ATTAACCCTCACTAAAGGGACTAG to CCCTT

  36. Pseudoknots

  37. Hammerhead Ribozyme

  38. tRNA Secondary Structure

  39. RNA Tertiary Structure

  40. Pyruvate Kinase

  41. Human DNA clamp PCNA

  42. Chou-Fasman Parameters

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