Protein Analysis Tools 2 - PowerPoint Presentation

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Protein Analysis Tools 2 - PPT Presentation

nd April 2012 Ansuman Chattopadhyay PhD Head Molecular Biology Information Service Health Sciences Library System University of Pittsburgh ansumanpittedu httpwwwhslspitteduguidesgenetics ID: 672996

sequence protein pitt media protein sequence media pitt hsls http clc main alpha mahagrtgydnreivmkyihyklsqrgyewdagdvgaappgaapapgifssqpghtphpaasrdpvartsplqtpaapgaaagpalspvppvvhltlrqagddfsrryrrdfaemssqlhltpftargrfatvveelfrd gvnwgrivaffefggvmcvesvnremsplvdnialwmteylnrhlhtwiqdnggwdafvelygpsmrplfdfswlslktllslalvgacitlgaylghk alignment ref lymphoma isoform

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Slide1

Protein Analysis Tools2nd April, 2012

Ansuman Chattopadhyay, PhD, Head Molecular Biology Information ServiceHealth Sciences Library SystemUniversity of Pittsburghansuman@pitt.eduhttp://www.hsls.pitt.edu/guides/genetics Slide2

What we’ll do:Brief overview of CLC Main Workbenchfind genomic context of a protein sequencesearch for the presence of conserved domainscreate a multiple sequence alignment plotSlide3

What we’ll do:analyze primary structure such as, hydrophobicity, hydrophylicity, antigenicity

, repeat sequence detection etc. predict secondary structurepredict post translational modification such as, Phosphorylation, glycosylation, ….search for interacting partnerspredict domain driven

protein-protein interactionsSlide4

Workshop Resources

http://www.hsls.pitt.edu/molbio/tutorialsSlide5

HSLS MolBio VideosSlide6

Sequence Analysis Software SuitsWisconsin GCGVectorNTIDNA STAR-LaserGeneGeneiousCLC MainSlide7

Why CLC Main ?WindowsMacLinuxDNA, RNA, Protein, Microarray Data AnalysisRegular UpdateHSLS LicensedSlide8

CLC Main AccessHSLS CLC Main RegistrationLink: http://www.hsls.pitt.edu/molbio/clcmainAccess via Pitt - Network ConnectInstruction video: http://goo.gl/JNjMtSlide9

CLC Main Workbench OverviewGraphical Users InterfaceProtein sequences ImportSequence NavigationSlide10

CLC Main Graphical User Interface (GUI)Slide11

CLC MainSlide12

Navigate a proteinsequenceSlide13

CLC Main –getting started (basic navigation steps): http://media.hsls.pitt.edu/media/molbiovideos/clc-navigation-ac0312.swfCLC Main Workbench Walkthrough (Part1): http://media.hsls.pitt.edu/media/molbiovideos/clcmain-walkthrough-part1-ac0112.swfCLC Main Workbench Walkthrough (Part2): http://media.hsls.pitt.edu/media/molbiovideos/clcmain-walkthrough-part2-ac0112.swf

VideosSlide14

Import a Protein SequenceSlide15

Protein SequenceHuman PLCg1Refseq no: NP_002651Uniprot Accession Number: P19174FASTA fileRaw sequenceCLC features:

Search, Import, Create new sequenceSlide16

Import a DNA /Protein sequence into CLC Main (Part1):http://media.hsls.pitt.edu/media/molbiovideos/clc-import-part1-ac0112.swfImport a DNA /Protein sequence into CLC Main (Part 2):http://media.hsls.pitt.edu/media/molbiovideos/clc-import-part2-ac0112.swf

VideosSlide17

CLC protein sequenceSlide18

Protein sequence manipulationCreate a new protein with PLCg1 SH2-SH2-SH3 domainsSlide19

Sequence Alignment Pair-wise AlignmentGlobalLocal

Multiple Sequence AlignmentSlide20

Sequence AlignmentSlide21

Pair-wise Sequence AlignmentSlide22

Multiple Sequence AlignmentSlide23

Multiple Sequence AlignmentTools: ClustalW and T-coffeeSlide24

PLCg1 Orthologous sequencesPLCg1:Mouse: NP_067255Rat: NP_037319Cow: NP_776850Dog: XP_542998Zebra fish: NP_919388

Human: NP_002651NP_067255,NP_037319,NP_776850,XP_542998,NP_919388,NP_002651Slide25

Create a multiple sequence alignment plot using CLC(part1):http://media.hsls.pitt.edu/media/molbiovideos/msf-clcmain-ac0212 part1.swfCreate a multiple sequence alignment plot using CLC (part2):http://media.hsls.pitt.edu/media/molbiovideos/msf-clcmain-ac0212-part2.swfCreate a multiple sequence alignment plot: http://media.hsls.pitt.edu/media/clres2705/msa.swf

Compare two peptide sequences.: http://media.hsls.pitt.edu/media/clres2705/blast2.swfVideosSlide26

Starting with a short peptide sequence find:the whole protein sequenceorthologs in other species (nematode)Tool:UCSC BLATNCBI BLAST against SwissProtSlide27

Peptide to whole proteinPeptide seq: SPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPRSlide28

Place a mRNA or peptide sequence into the human genome (BLAT):http://www.hsls.pitt.edu/molbio/videos/play?v=12eFind homologous sequences: http://media.hsls.pitt.edu/media/clres2705/blast.swf

VideosSlide29

Find homologous sequence

SPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPRSlide30

Sequence Manipulation & Format ConversionSequence Manipulation Suitehttp://bioinformatics.org/sms2/Readseqhttp://thr.cit.nih.gov/molbio/readseq/

GenePept

FASTASlide31

Hands-OnRetrieve amino acid sequence present between position 25 to 45 in Sequence A (MS Word Doc)Identify the rat gene which encodes this peptide fragment and retrieve its whole protein sequenceFind the fruit fly

homolog of this protein.What % identity the fruit fly protein shares with its rat homolog?Predict potential MAPK phosphorylation sites present in the fruit fly proteinSlide32

Protein Domain Search: InterPro ScanInterPro is a database of protein families, domains, regions, repeats and sites in which identifiable features found in known proteins can be applied to new protein sequences.

>gi|72198189|ref|NP_000624.2| B-cell lymphoma protein 2 alpha isoform MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRD GVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHKSlide33

Videos:Find protein domains, PTM, secondary str etc: http://media.hsls.pitt.edu/media/clres2705/uniprot.swf Start with a protein pattern and find what proteins posses that domain: http://media.hsls.pitt.edu/media/clres2705/scanprosite.swf

Search for protein domains,repeats and sites: http://media.hsls.pitt.edu/media/clres2705/interpro.swfSlide34

Protein Domain Search: ScanProsite>gi|72198189|ref|NP_000624.2| B-cell lymphoma protein 2 alpha

isoform MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRD GVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHKSlide35

Pattern Search [AC]-x-V-x(4)-{ED}:This pattern is translated as: [Ala or Cys]-any-Val-any-any-any-any-{any but Glu or Asp}F-[GSTV]-P-R-L-[G>]Slide36

Pattern SearchSlide37

Protein Primary Structure AnalysisTool: ExPASy from SIBCalculated Mol WtTheoritical PIExtinction coefficients

Estimated half-lifeHydropathicity plot : Kyte & Doolittle Hydrophilicity plot: Hopp T.P., Woods K.RSlide38

Antigenic Site PredictionTool: Emboss Antigenic

EmBoss AntigenicAntigenic predicts potentially antigenic regions of a protein sequence, using the method of Kolaskar and Tongaonkar.Analysis of data from experimentally determined antigenic sites on proteins has revealed that the hydrophobic residues

Cys, Leu and Val, if they occur on the surface of a protein, are more likely to be a part of antigenic sites. A semi-empirical method which makes use of physicochemical properties of amino acid residues and their frequencies of occurrence in experimentally known segmental epitopes was developed by Kolaskar and Tongaonkar to predict antigenic determinants on proteins. Application of this method to a large number of proteins has shown that their method can predict antigenic determinants with about 75% accuracy which is better than most of the known methods. This method is based on a single parameter and thus very simple to use.Slide40

Transmembrane Region predictionSlide41

Transmembrane Site PredictionTool: TMHMM Server

Protein Secondary Structure

>gi|72198189|ref|NP_000624.2| B-cell lymphoma protein 2 alpha

isoform

MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRD GVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHKSlide43

Protein-Protein Interactions PredictionTool: STRING

Hands-onTake the human BCL2 protein sequence and Find its domain architecturePredict the topology of its transmembrane regionDesign suitable antigenic site for antibody generationWhat is its calculated Mol Wt and Ext Coefficient?

Predict its secondary structureWhat % of this protein possesses alpha helical structure?Predict its potential interacting partnersSlide45

Hands-onPrediction of potential phosphorylation sites present in a protein sequence.Sequence: human BCL2>gi|72198189|ref|NP_000624.2| B-cell lymphoma protein 2 alpha isoform MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRD GVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHKSlide46

Phosphorylation Site Prediction:

Tool:

NetPhosSlide47

Phosphorylation Site Prediction:

Tool: GPSSlide48

Thank you!Any questions?Carrie Iwema Ansuman Chattopadhyay

iwema@pitt.edu ansuman@pitt.edu 412-383-6887 412-648-1297http://www.hsls.pitt.edu/guides/genetics