proteomics in detail Power point presentaion.ppt

Proteomics

1. -Presented by:
Peter Oledzki
John Pinney
Ashwin Sivakumar

2. Proteomics has been said to be the next step from genomics
Proteomics is the sudy of the proteome.
The proteome is the complete complement of proteins found
in a complete genome or specific tissue.
Proteomics

3. Proteomics and genomics are inter-dependent
Genome Sequence
mRNA
Primary Protein products
Functional protein products
Determination of gene
Genomics
Proteomics
Proteomics
Protein Fractionation
2-D Electrophoresis
Protein
Identification
Post-Translational
Modification

4. Aims of Proteomics
Detect the different proteins expressed by
tissue, cell culture, or organism using 2-
Dimensional Gel Electrophoresis
Store those information in a database
Compare expression profiles between a
healthy cell vs. a diseased cell
The data comparison can then be used for
testing and rational drug design.

5. Gel Electrophoresis
Motion of charged molecules in an electric
field.
Polyacrylamide gel provides a porous matrix
–(PAGE –Polyacrylamide Gel Electrophoresis)
Sample is stained with comassie blue to
make it visible in the gel.
Sample placed in wells on the gel.

6. 1-D Gel electrophoresis
Separation in only
1 dimension: size.
Smaller molecules
travel further
through the gel
then large
molecules, thus
separation.

7. 1-D continued
Electric field across gel separates molecules.
–Negatively charged molecules travel towards the
positive terminal and vice-versa.
–Western blotting(Protein) not to be confused with Southern
blotting (DNA) or Northern blotting (RNA)
Proteins are treated with the denaturing detergent
SDS (sodium dodecyl sulfate) which coats the protein
with negative charges, hence SDS-PAGE.

8. 2-D –Separation is based on size
and charge
First step is to separate based on charge or
isoelectric point, called isoelectric focusing.
Then separate based on size (SDS-PAGE).

9. Isoelectric Focusing
The isoelectric pointis the pH at which the
net charge of the protein molecule is neutral.
Different proteins have different isoelectric
points.
Isoelectric point is found by drawing the
sample through a stable pH gradient.
The range of the gradient determines the
resolution of the separation.

10. SDS-PAGE
Second Dimension.
Separation by size.
Run perpendicular to Isoelectric focusing.
The only unresolved proteins after the first
and second dimensions are those proteins
with the same size and same charge –rare!

11. 2-D Proteomics Example

12. 2D-PAGE Analysis Software
2D-PAGEtechnologyhasbeeninuseforover20
years,andpotentiallyprovidesavastamountof
informationaboutaproteinsample.
However,duetodifficultieswithdataanalysis,it
remainsonlypartiallyexploited.

13. Analysis problems
Itcanbeverydifficulttocomparetheresultsoftwo
experimentstoyieldadifferentialexpressionprofile:
Canbeseverewarpingofgeldueto
–unevencoolantflow
–voltageleaks
–tearsingel
Canbeproblemswithnormalisationof
–background
–spotintensity
Canbedifferencesinsamplepreparations.

15. Current state of software
Correctidentificationandalignmentofspotsfromthe
twogelshasgenerallybeenaprocesswithalotof
manualintervention-henceveryslow.
Theprocessingpoweravailablewithtoday’sPCs
meansthatautomatedanalysisisstartingtobecome
possible.
Onevendorclaimsathroughputof4gelpairsper
hourcanbecomparedandannotatedbyan
experienceduseroftheirpackage.

16. Automated gel matching
Gelmatching,or“registration”,istheprocessof
aligningtwoimagestocompensateforwarp.
Somepackagesstillrequiretheusertoidentify
correspondingspotstohelpwithgelmatching.
TheZ3programfromCompugenhasafully-
automatedgelmatchingalgorithm:
–definesetofsmall,uniquerectangles.
–computeoptimallocaltransformationsforrectangles.
–Interpolatetomakesmoothglobaltransformation.
Notethatthismakesuseofspotshape,streaks,
smearsandbackgroundstructure,whichother
programsdiscard.

20. Spot detection
Oncethegelimageshave
beenmatched,theprogram
automaticallydetectsspots.
Algorithmsaregenerally
based on Gaussian
statistics.

21. Spot Quantitation
Thepositionsofdetectedspotsarecalibratedtogive
apI/mWpairforeachprotein.
Avaluefortheexpressionleveloftheproteincanbe
calculatedfromtheoverallspotintensity.
Someprogramsdonotquantitateeachgel
separately,butcalculaterelativeintensitypixelby
pixel.Thismaybeamoreaccurateapproach.

22. Differential Expression
Theusercansetthreshold
valuesforthedetectionof
differentialexpression.This
helpsreducetheamountof
informationdisplayedat
once.
Inthisexample,aprotein
expressedonlyinthe
secondsampleiscircledin
red.Theyellowcirclesshow
proteins which are
differentiallyexpressed.

23. Annotation
Somesystemsallowsemi-automaticannotationof
spots,basedonadatabaseofproteinslistingtheir
pI/mWvalues.
Proteinsofinterestcanalsobeexcisedfromthegel
andsentontomassspectrometryfordefinitive
identification.TheProteomeWorkssystemfromBio-
radofferssuchanintegratedsolutionfor2D-PAGE
andMALDI.

24. Multi-experiment Analysis
Oneusefulfeatureofmodernprogramsistheability
tocollatedatafrommanyrunsofthesame
experiment.
Spotswhichonlyappearinonegelarelikelytobe
artifacts,andareremovedfromtheanalysis.
Thisisanexcellentwaytoreducenoiseandenhance
weaksignals.

25. Links
Z3 system (Compugen) -http://www.2dgels.com/
Melanie3 (SIB) -http://us.expasy.org/melanie/
ProteomWeaver (Definiens) -
http://www.proteomweaver.com/
PDQuest (Bio-Rad) -http://www.biorad.com/
Delta2d (Decodon) -http://www.decodon.com/

26. Introduction to the databases
•Withtheadventofmany2-DPAGEdatabasestherearea
numberofproteinspotsthatarealready"identified"inafew
celllines.Combinedwiththeaimsoftheexperiment,these
databasesmaygiveonetheopportunitytoguessatthe
identityofaparticularproteinspotandconfirmordenythis
byimmunoblotting.Theapproachofobtainingaccurate
peptidemassesfromspecificallycleavedproteinstosearch
proteinsequencedatabases,knownaspeptidemass
fingerprinting,providesonewithanotheropportunityto
identifyapreviouslysequencedproteinor(hopefully)
confirmthatitisindeednovel.
An animated SDS PAGE presentation

27. •Anumberof2-DGeldatabasesexist.
•Quantitativedatabases:S.cervisiaeand
REF52.
•Annotativedatabases:E.coliandhuman
keratinocytes.
•Anannualissueofthejournal
“Electrophoresis”-Majordatabaseforthese
databases!!!…(Imeanhaslinkstomanyof
these).
•Abestonewouldobviouslythedatabase
whichisregularlyupdated.(Eg:Swiss2D
page).

28. List of 2-D GEL DATABASES
One can find an extensive list of such databases by following
these links.
We would discuss a few “Interesting ones”.
•World 2-D PAGE
•NCIFCRF
•DEAMBULUM -Protein Databases
•Ludwig Institute of Cancer Research
•Phoretix

29. World 2-D Page:Index of 2-D page Databases-ExPaSy
•Basicallyalinktovarious2-DPagedatabases.
•Hasausefultoolcalled2-DHuntwhereonecould
searchfor2-DErelatedsitesontheweb.
•Indexedasdatabasesformultispecies,mammalia,yeast,
plant,bacteria,virusesandparasites,celllines.

30. Swiss 2-D Page
•Basicallyaproteindatabankfor2-DpageandSDSpagereference
maps.
•Maygivetheexactlocationoftheproteininthemaportheregionin
themapassumingthefactthatithasaSwissprotentry.
•Options:Searchbykeywords,Accessionnumber,spotclicking,full
text,author,Swiss-2DPagespotserialnumber,SRS.Mostofthembeing
self-explanatory.
•Proteinlistforaparticularreferencemap(table)(canbe
downloaded).Itgivesdetailsonthegenename,proteindescription,S-
2DPreferencenumber,S-2DPaccessionnumber,identification
method,Exp.MolecularweightsandPisforeachentityfound.
•Wecanalsolocatethelocationofaproteinsequencein
all/one/selectedreferencemapsavailable.Ifitisnotfoundatemporary
virtualentryiscreatedontheExPASyserver.

31. SWISS 2D-PAGE (contd)
•ItgivescrossreferencetoMedlineandafewother
databases.
•Inadditiontothistextualdata,SWISS-2DPAGEprovides
several2-DPAGEimagesshowingtheexperimentally
determinedlocationoftheprotein,aswellasatheoretical
regioncomputedfromthesequenceprotein,indicating
wheretheproteinmightbefoundinthegel.
•Genbio(GenevaBioinformatics)givessubscription(PAID)
fortheSwiss2DPAGEtoCommercialInstitutions.
•VitalStatistics
¯Currentrelease(15.0)has861entriesin33referencemaps.

32. ¯Vitalstatscontinued...
¯Sourcesofreferencemaps:
¯Human(Liver,plasma,HepG2,RBC,Lymphoma,HepG2Secreted
Proteins,CBF,MacrophagelikeCellLine,Erythroleukemiacell,platelet,
kidney,promycelocyticleukemiacells,colorectalepitheliacells,
colorectaladenocarcinomacellline(DL-1),Solublenuclearproteinsand
matrixfromlivertissue)
¯Mouse(Liver,gastrocnemiusmuscle,pancreaticisletcells,brown
adiposetissue,whiteadiposetissue,solublenuclearproteins,matrixfrom
livertissue).
¯Arabidopsisthaliana
¯Dictyosteliumdiscoideum
¯Escherichiacoli(for7pIranges:3.5-10,4-5,4.5-5.5,5-6,5.5-6.7,6-9,6-
11)
¯Saccharomycescerevisiae

33. Swiss 2D Page(cont..)
Therehavebeensomerecentadditionstothedatabase.
SDSand2-DPageofnuclearproteinsfromHumanHeLa
cellshavebeenaddedtothegrowinglistofreference
maps.Itisstillanongoingproject.Informationaboutknown
proteinsfoundwithinthatgelstretchhasbeenmapped(see
beloe:right-SDS,left-PAGE)

34. Swiss2DPage(cont)
SomeUsefulabbreviations:
-IDline:comprisesofID,Entryname,Entryclassandthe
method(2Dgel)intheorderasmentioned.Theyfollowa
specificnomenclature.
-AClinebasicallycontainsAccessionnumbersseperated
byasemicolon.It’sastablewayofidentifyingentries
witheachrelease.
-DTLinespecifiesdate(selfexplanatory!).
-DELinegivesadescriptiveinformationaboutthe
protein.Ifthecompletesequencewasnotdeterminedthen
lastlinewouldspellas“Fragment”.

35. Someusefulabbreviations(cont…)
-TheIMline
TheIM(Images)lineslistthe2-DPAGEandSDS-
PAGEimageswhichareassociatedtotheentry.These
maybe,forexample,TUMORALLIVER,NORMAL
LIVERorjustLIVER.
-RP(ReferencePosition)line:Describesextentofwork
carriedoutbytheauthor.Eg:Proteinsequence,amino
acidcomposition,mappingongel,characterisationand
review.
-The “O” seriescontainsorganism
species(OS),taxonomy(OX)andclassification(OC).
-MT(Master)linehasinformationabouttypesofmaps
used(Eg:Plasma,liveretc).

36. Methodsusedforzeroingontheidentifiedspots.
•Totalof3398identifiedspots(asofthelatest
version).
•Aminoacidcompositionhasidentified5.3%of
thesespots.
•Co-migration:2.6%
•Gel-matching:46.7%
•Immunoblotting:20%
•Microsequencing:15.5%
•Peptidemassfingerprinting:26.3%
•Tandemmassspectroscopy:2.3%
Well..does it carry
a message?

37. Browsing the Swiss 2-D Page using spot clicking
-Wecouldgetinformationaboutaknownproteinbyclicking
ononeofthe“checks”intheextensivelistofimagemaps
available.
-Onclickingitthrowsatailor-readyimagemapshowingthe
accurate/approximatepositionofthatproteinwithrespectto
alltheimagemapsavailable.Butforobviousreasonsthebest
viewcanbeobtainedfromthereferenceimagemapwe
initiallyclicked.
-AhypertextlinkcanthenbeusedtoobtainthefullSWISS-
PROTentryforthatprotein,displayingproteinsequence,
domainstructure,informationonknownpost-translational
processingandmodifications,andreferences.

38. Image clicking(continued…)
-FromSWISS-PROT,theusercanselectalinkto
SWISS-3DIMAGE toseethethree-dimensional
structureoftheprotein,ifitisknown,ortosubmitthe
sequencetotheSWISS-MODELthree-dimensional
modellingtoolorviewthedomainstructure.
-Also,fromSWISS-PROT,theusercanselectlinksto
pertinentinformationfromDNAsequencedatabases
(EMBL/Genbank),chromosomalandgenomicmaps
(GDBGenomeDatabase),bibliographicreferencesand
abstracts(Medline),anddatabasesontheassociationof
humanproteinswithdiseases(OMIMOnline
MendelianInheritanceinMan).

39. Diagramatic representation of Image
Clicking... Here we click on
this spot in
reference map of
the Colorectal
epithelia cell
Throws a screen
showing
the pictures of
different image
maps with respect
to that protein

40. Diagramatic representation(cont…)
Protein
identification
on chosen reference map The red
rectangle is the
expected region
of the protein
on the gel.
Spots are the
proteins
identified
Dotted lines are extensions of
the possible regions if the
protein is acetylated,
phosphorylated or
glycosylated.

41. Enough of Swiss 2D PAGE!!!
-Onimageclickingwecouldalsocalculatethe
theoreticalpIandMolecularweightofdifferent
sequencefragmentswithdesiredendpoints.One
couldspecifytheN-TerminalandC-Terminal
valuesintheoptionsavailableinthescreen.By
defaultitwouldcomputeitfortheentiresequence
available.
-Swiss2Dpagealsohasacrossreferenceto
anotherpopular2DGeldatabaseinSiena2DGel
database.
Nowbyebye!Swiss2DPAGE.

42. Biobase/Julio Celis Database
(Very well structured & lucid!!!)
Preparation and labelling of Human keratinocytes-please visit:
http://biosun.biobase.dk/~pdi/procedures/procedure_label.html
-Hosted at Danish Center of Human Genome Research.
-Have the distinction of constructing the first 2D Gel database(HeLa cells) in
1981(Bravo, R., Bellatin, J. and Celis, J.E).
-Human and Mouse 2D PAGE Databases.
-annotated 2-D gel pattern of fluids from different species can be found in the
fluid gallery.
-One can find 2D-Gel immunoblots of selected proteins against various
antibodies.
-2D Gel gallary of various human cell types and fluids.(Includes tumors,
keratinocytes and post-translational modifications.).

43. Biobase/Julio Celis Database(cont…)
Human 2-D PAGE Database:
Thekeratinocyte2DPAGEdatabaseconstructedusingcarrier
ampholytes,isthelargestofitskindandcurrentlylist3625
cellular(2313isoelectricfocusing,IEF;954nonequilibrium
pHgradientelectrophoresis,NEPHGE),andexternalised
polypeptides(358,IEF)ofwhich1285havebeenidentified
usingacombinationoftechniquesincludingimmunoblotting
[32],Edmandegradationofinternalpeptides[33,34],andmass
spectrometry[35].(Mightbeoutdated!!!!)
Representation through flow charts to
follow...

44. (Biobase Cont..)
By clicking on each of the available reference gels,we can get
information(links to medline,swissprot,PDB,cellular
location,Knockout,method used) on the available
proteins(checked spots) on the gel.
Databases for study of skin biology
HK-IEF d’baseHK-NEPHGE d’base
KP present in medium
IEF Database
865 IP 372 IP
59 IP

45. Biobase(cont…)
Database for study of Bladder Cancer
TCC-IEF database
TCC-NEPHGE d’base
BSCC-IEF d’base
Urine-IEF d’base
449 IP
144 IP
309 IP
197 IP

46. Biobase(cont…)
Other 2D Page Databases
Human MRC-5-
Fibroblasts-IEF
D’base
Human MRC-5-
Fibroblasts-NEPHGE
D’base
262 IP 84 IP

47. Biobase(cont…)
Search Options:
Seacrh by protein name, keyword, sample spot number,
RelativeMolecular mass, pI, organelle /component.
Other options relating listing of proteins,views of the gels are
quite self explanatory.
Other utilities of the Database:
Has links to
-NCBI’S Human-Mouse Homology maps through its Mouse
2D-PAGE Databases.
-Interesting studies like Mouse-Genome Informatics(Jackson’s
lab) and Mouse Atlas Projects.

48. NCIFCRF(National Cancer Institute…..could
not sphere out what FCRF was!!!..sorry)
*Seems a very exhaustive and useful source.Lots of things
still to study.*
2D Protein Gel Databases
Maintained by
Image Processing
Section
Maintain the
gel analysis
software-
GELLAB II
WebGel Flicker dbEngine

49. WebGel:
WebGel is an Internet-based, interactive, qualitative and
quantitative gel
database analysis system.
A WebGel database contains previously quantified gel data
generated from a
stand-alone quantitataive gel analysis system.
wbdemoDB
demonstration
database
of serum
proteins in a
fetal alcohol
syndrome study.
melanie2DB
demonstration
database
of E.coli gelsfrom the
Melanie 2.3
demonstration
database.
fasDB
database
of serum proteins
in a fetal alcohol
syndrome study

50. Flicker comparing two Plasma 2D-PAGE Gels.
FLICKER
“Flicker is a method for comparing images from different
Internet sources on your Web browser. In the case of 2D
protein electrophoretic gel images, maps identifying proteins in
these gels are becoming increasingly available. Visually
comparing 2D sample gels against these 2D gel database maps
may suggest putative protein spot identification in many cases.
Flicker was originally developed for comparing 2D protein
gels across the Internet.”
-Part of the description in their web site.

51. dbEngine
It is a simple database search engine which may be used to
quickly create a searchable database on a World Wide Web
(WWW) server. Data may be prepared from spreadsheet
programs (such as Excel, etc.) or from tables exported from
relational database systems. This Common Gateway Interface
(CGI-BIN) program is used with a WWW server such as
available commercially, or from NCSA or CERN. Its
capabilities include: 1) searching records by combinations of
terms connected with ANDs or ORs; 2) returning search results
as hypertext links to other WWW database servers; 3) mapping
lists of literature reference identifiers to the full references; 4)
creating bidirectional hypertext links between pictures and the
database.
Manual

52. SIENA 2-D PAGE
-Similar to Swiss 2D PAGE when it comes to
browsing the Database.
-Point to be noted: Last update was June 2000.
-Their Gel entries include many human protein
maps.
-For further information please toy around with their
site!

53. PROTEOME Inc.
-Available databases are:
-Caenorhabditis elegansProteome Database(YPD)
-Saccharomyces cerevisiaeProteome Database (WormPD)
-S.pombe (PombePD)
-Human PSD(Quite interesting!!!):
Its sort of a survey database.It has greater than 17,000
human, mouse and rat proteins.Within this their
PDtm(Protein Coupled Receptor database) has around
600 GPCRs.
As with the case with most companies(Trick up their
sleeve!)…their Human PS database is available only for
subscription.

54. HSC-2D PAGE (Harefield)
-Hosted at Heart Science Center at Harefiled.
-Individual gel databases available:
Human Heart
Human Endothelial cell
Rat Heart
Dog Heart
-Well..visit their site,again quite similar to the Swiss 2D-
PAGE.
Ventricles

55. PPMDB at Sphinx
•Warning!!!“Itsnomoreupdated”….butcouldstillbea
usefulsource.
•WasaimedtocreatedirectoryofArabidopsisplasma
membraneproteinsandtoobtainexpressionandsequence
data.
•Includes->
•a2-Dmapagreeingtotherulesoffederated2-Ddatabases
•arepertoireofplasmamembraneproteinsnot-presentonthe
map
•proteinsequencedatalinkedtoESTorcDNAdata
•andproteinexpressiondataaccordingtoecotypesandplant
organs.

56. Sphinx(cont…)
-AvailablereferencegelsincludeCallus2Dgels,Cationicand
Anionicdetergent2Dgels,analyticalplantorgans,analytical
solubizationmethods,analyticalecotypesandpreparative.
-OnecouldqueryasequenceagainstPPMdbsequencestofind
matches.
-Onclickingonsequencematchingwecanfinddetailed
informationaboutprocedureslike2DE,Running,stainingand
scanning.
-Wecouldalsoperformamoreelaboratesearchonavailablegels
byenteringuseroptionslikeproteinname,sub-cellular
location,accessionnumber,hydrophobicityproperties,Gel
family,MW,pIetc.

57. Challenges/shortcomings in 2D-Gel Databases
(Food for thought!)
-Detectionofverylowabundancypolypeptides.
-Theresolutionofverybasicandhighmolecularweight
proteins
-Thelackofsatisfactoryquantitationproceduresforthe
analysisofalltheproteinsresolvedinthegels.
-Storageofimageanalysisresults
-Dataintegrity-Mergingdatafromdifferentsources?
-Explanatorynotesonthereferencemaps?
Subscribetoswiss-flashnewslettertokeep
yourselfupdatedon2Dgelandotherresources
availabletoExPASy!!!

58. Try these links
•http://www.2dgels.com/
•http://www.genomicglossaries.com/content/lifesciences_data
basesdirectory.asp
•http://www.expasy.ch/ch2d/2d-index.html
•http://www.infobiogen.fr/services/deambulum/english/db4.ht
ml#GELS2D
•http://www.bio-mol.unisi.it/2d/2d.html
•http://biobase.dk/cgi-bin/celis
•http://www.phoretix.com/customers/wl_2d_specific_sites.ht
m
•http://sphinx.rug.ac.be:8080/ppmdb/index.html
and thenTime to chill!!!