intro
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IntroSpring 2009 Bioinformatiatics
Proteomics
workflowSpring 2009 Bioinformatiatics
Proteomics Workflow• Sample Prep• Sequencing• Database Search• Protein ID• Protein Interactions
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
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
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
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
TemplateSpring 2009 Bioinformatiatics
FractionationSpring 2009 Bioinformatiatics
Affinity Purification
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)
Unusual amino acidsSpring 2009 Bioinformatiatics
Unusual Amino Acids
phosphorylationSpring 2009 Bioinformatiatics
Phosphorylation - signal transduction
mRNA
mRNA
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MS analysis
Antibody arrays
Good for low-abundance proteinsProblem is antibody specificity
Array-based protein interaction detection
Protein microarrays
Yeast Two-Hybrid System
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
Interaction maps - Grid
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
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
IntroSpring 2007 Bioinformatiatics
RNA and Protein Structure Prediction
SSU Secondary StructureSSU Secondary Structure
Ribosome
Ribosome
-Beaudry & Joyce, Science, 1992
Frq. Of mutation
(%; n=25) after
9 generations.
M13 vector sequence
TATAGGGCGAATTGAATTTAGCGGor
ATTAACCCTCACTAAAGGGACTAG
to
CCCTT
Pseudoknots
Hammerhead Ribozyme
tRNA Secondary Structure
RNA Tertiary Structure
Pyruvate Kinase
Human DNA clamp PCNA
Chou-Fasman Parameters
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