Intro Spring 2009 Bioinformatiatics Proteomics. workflow Spring 2009 Bioinformatiatics Proteomics Workflow Sample Prep Sequencing Database Search Protein.

IntroSpring 2009 BioinformatiaticsBioinformatiatics

Proteomics

workflowSpring 2009 BioinformatiaticsBioinformatiatics

Proteomics Workflow

• Sample Prep• Sequencing• Database Search• Protein ID• Protein Interactions

Identification

Quantification

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

PMF

MS/MS

DIGE

LC-MS & Tags

Sequence data bases:EMBL Nucleotide Sequence Database GenBank UniProtKB/Swiss-Prot & TrEMBL Ensemble EST database PIR

Identification

Quantification

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

data

MSight -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 BioinformatiaticsBioinformatiatics

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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FractionationSpring 2009 BioinformatiaticsBioinformatiatics

Affinity Purification

2D gels at SwissprotSpring 2009 BioinformatiaticsBioinformatiatics

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 BioinformatiaticsBioinformatiatics

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 BioinformatiaticsBioinformatiatics

Unusual Amino Acids

phosphorylationSpring 2009 BioinformatiaticsBioinformatiatics

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 BioinformatiaticsBioinformatiatics

RNA and Protein Structure Prediction

SSU Secondary StructureSSU Secondary StructureSSU Secondary StructureSSU Secondary Structure

Ribosome

Ribosome

-Beaudry & Joyce, Science, 1992

Frq. Of mutation

(%; n=25) after

9 generations.

M13 vector sequenceM13 vector sequence

TATAGGGCGAATTGAATTTAGCGG

or

ATTAACCCTCACTAAAGGGACTAG

to

CCCTT

PseudoknotsPseudoknots

Hammerhead RibozymeHammerhead Ribozyme

tRNA Secondary StructuretRNA Secondary Structure

RNA Tertiary StructureRNA Tertiary Structure

Pyruvate KinasePyruvate Kinase

Human DNA clamp PCNA

Chou-Fasman ParametersChou-Fasman Parameters