intro
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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IntroSpring 2009 Bioinformatiatics
Proteomics
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workflowSpring 2009 Bioinformatiatics
Proteomics Workflow• Sample Prep• Sequencing• Database Search• Protein ID• Protein Interactions
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IdentificationQuantification
General workflow of proteomics analysis
External data sourcestaxonomy, 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
PMFMS/MSDIGELC-MS & Tags
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Sequence data bases:EMBL Nucleotide Sequence Database GenBank UniProtKB/Swiss-Prot & TrEMBL Ensemble EST database PIR
IdentificationQuantification
General workflow of proteomics analysis
Proteins/peptides
Digestion and/or separation
MALDI, MS/MS
2D Page data basesSwiss 2D PAGE, Gelbank, Cornelia, WordPAGE
Make 2D
Imaging tools:Melanie, PDQuest ProgenesisDelta 2D
Storing/ organising:ProteincsapeMSight
KEGG PDB DIPOMIMReactomePROSITPfamSPINBONDSTRINGAmiGODavidPubMedMEDLINE
MascotSequestAldentePopitamPhenyxFindModProfoundPepFragMS-FitOMSSASearch XLinksTagIdent
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General workflow of proteomics analysis
Proteins/peptides
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 2DProteinscape –platform for storing, organizing
dataMSight -representation of mass spectra along with data from the separation
2D gel databases:Data integration on the webImage data and textual information
•Swiss 2D PAGE •Gelbank •Cornelia•WordPAGE
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Laser capture
Spring 2009 BioinformatiaticsLaser-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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TemplateSpring 2009 Bioinformatiatics
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FractionationSpring 2009 Bioinformatiatics
Affinity Purification
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2D gels at SwissprotSpring 2009 Bioinformatiatics
Swissprot ExPaSy Database
2D Electrophoresis
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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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codon UsageSpring 2009 Bioinformatiatics
Standard Genetic Code (transl_table=1) AAs = FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGGStarts = ---M---------------M---------------M----------------------------Base1 = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGGBase2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGBase3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG
AAs = FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGGStarts = ---M---------------M------------MMMM---------------M------------Base1 = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGGBase2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGBase3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG
The Bacterial and Plant Plastid Code (transl_table=11)
AAs = FFLLSSSSYYQQCC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGGStarts = -----------------------------------M----------------------------Base1 = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGGBase2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGBase3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG
The CiliateHexamita Nuclear Code (transl_table=6)
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Unusual amino acidsSpring 2009 Bioinformatiatics
Unusual Amino Acids
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phosphorylationSpring 2009 Bioinformatiatics
Phosphorylation - signal transduction
mRNA
mRNA
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MS analysis
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Antibody arrays
Good for low-abundance proteinsProblem is antibody specificity
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Array-based protein interaction detection
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Protein microarrays
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Yeast Two-Hybrid System
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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
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Interaction maps - Grid
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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
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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
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IntroSpring 2007 Bioinformatiatics
RNA and Protein Structure Prediction
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SSU Secondary StructureSSU Secondary Structure
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Ribosome
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Ribosome
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-Beaudry & Joyce, Science, 1992
Frq. Of mutation
(%; n=25) after
9 generations.
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M13 vector sequence
TATAGGGCGAATTGAATTTAGCGGor
ATTAACCCTCACTAAAGGGACTAG
to
CCCTT
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Pseudoknots
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Hammerhead Ribozyme
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tRNA Secondary Structure
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RNA Tertiary Structure
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Pyruvate Kinase
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Human DNA clamp PCNA
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Chou-Fasman Parameters
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