Protein and nucleic acid sequencing

Protein Sequencing
Introduction
Protein Sequencing
History of Protein Sequencing
Determining Amino Acid Composition
N-terminal amino acid analysis
C-terminal amino acid analysis
Edman degradation
The Edman degradation reaction
Limitations of the Edman degradation
Mass spectrometry
Nucleic Acid Sequencing
Introduction
Nucleic Acid Sequencing
Type of Nucleic Acid Sequencing
DNA Sequencing
Method of DNA Sequencing
Application of DNA Sequencing
DNA Sequencing Institutes
Conclusion
Reference

1. By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. SYNOPSIS
Protein Sequencing
Introduction
Protein Sequencing
History of Protein Sequencing
Determining Amino Acid Composition
N-terminal amino acid analysis
C-terminal amino acid analysis
Edman degradation
The Edman degradation reaction
Limitations of the Edman degradation
Mass spectrometry

3. Synopsis
Nucleic Acid Sequencing
Introduction
Nucleic Acid Sequencing
Type of Nucleic Acid Sequencing
DNA Sequencing
Method of DNA Sequencing
Application of DNA Sequencing
DNA Sequencing Institutes
Conclusion
Reference

4. Introduction
Proteinsarelargebiologicalmolecules
consistingofoneormorechainsofaminoacids.
Proteinsdifferfromoneanotherprimarilyin
theirsequenceofaminoacids,whichisdictated
bythenucleotidesequenceoftheirgenes,and
whichusuallyresultsinfoldingoftheprotein
intoaspecificthree-dimensionalstructurethat
determinesitsactivity.

5. PROTEIN Sequencing
Proteinsequencing isatechniqueto
determinetheaminoacidsequence of
aprotein.
Proteinsareverylongpolypeptidechains
of100toseveralthousandaminoacid
residues.
Aminoacidscanbejoinedcovalently
throughpeptidebondstoformpeptides
andproteins.
Proteinsequencingdenotestheprocess
offindingtheaminoacidsequence,or
primarystructureofaprotein.

6. History
Thefirstproteinsequencingwasachieved
byFredericSangerin1953.Hedetermined
theaminoacidsequenceofbovineinsulin.
ForthisachievementSangerwasawarded
theNobelPrizein1958.
PehrEdman,developedamachine,
determiningpeptidesequencesautomatically,
calledaSequenator.

7. DETERMINIG AMINO ACID COMPOSITION
Essentialstepsforproteinsequencing
Separation
Hydrolysis
Hydrolysisisdonebyheatingasampleofthe
proteinin6MolarHClto100-110C°for24
hoursorlonger.
However,theseconditionsaresovigorousthat
someaminoacids(serine,threonine,tyrosine,
tryptophan,glutamineandcystine)are
degraded.

8. N-terminal Residue Identification
C –Terminal amino acid analysis
Edman degradation method
SEQUENCING METHODS

9. N-terminalResidueIdentification
Thisprocedureisoftenusedinconjugationwithhydrolysis
istolabel&identifytheaminoterminalaminoacidresidue.
ForthispurposeFredrickSangerdevelopedthereagent
1-fluoro-2,4dinitrobenzene(FDNB).
Aftertheaminoterminalresidueislabeled,thepolypeptide
ishydrolyzedtoitsconstituentaminoacidandthelabeled
aminoacididentified.
SEQUENCING METHODS

10. Someotherreagentsarealsousedtolabeltheamino
terminalresiduesuchasDansylchlorideandDabsyl
chloride.
Dansylchloridederivativesishighlyfluorescent
sulfonamidederivativesandcanbedetectedandmeasuredin
muchlowerconcentrationthandinitrophenylderivative.
Dabsylchloride,isnowcommonlyusedbecauseitforms
intensivelycolouredderivatives.

11. Sanger's method

12. C –Terminal amino acid analysis
ThemostcommonmethodistoaddCarboxypeptidase.
Carboxypeptidaseareenzymethatcleavesaminoacid
residuesfromC–terminusofpolypeptideinasuccessive
fashion.
AnothermethodofC–terminalaminoacidanalysisis
treatedwithhydrazine,whenapeptidegroupssplitandthe
carbonylcomponentisconvertedtocorresponding
hydrazine.

14. Edman degradation method
Tosequencetheentirepolypeptideachemicalmethoddevisedby
PehrEdmanisusuallyemployed.
TheEdmandegradationprocedurelabelsandremovesonlythe
amino-terminalresiduefromapeptidewithoutdisruptingtheother
peptidebondsbetweentheotheraminoacidresidues.
TheEdmandegradationsequentiallyremovesoneresidueatatime
fromtheaminoendofapeptide.
Phenylisothiocyanate(PTC)reagentisusedfortheEdman
degradation.

15. Thepeptideisreactedwithphenylisothiocyanate
undermildlyalkalineconditions,whichconverts
theaminoterminalaminoacidtoa
phenylthiocarbamoyl(PTC)adduct.
ThepeptidebondnexttothePTCadductisthen
cleavedinastepcarriedoutinanhydrous
trifluoroaceticacid,withremovaloftheamino-
terminalaminoacidasananilinothiazolinone
derivative.
Thederivatizedaminoacidisextractedwith
organicsolvents,convertedtothemorestable
phenylthiohydantoinderivativebytreatmentwith
aqueousacid,andthenidentified.

16. Edman Degradation method

17. LimitationofEdmandegradation
BecausetheEdmandegradationproceeds
fromtheN-terminusoftheprotein,itwillnot
workiftheN-terminalaminoacidhasbeen
chemicallymodified.
Only50aminoacidisidentifiedbythis
method,ifproteinchainislongerthan50
aminoacidthantheproteinchaincutby
differentmethod.

18. SEQUENCING OFLARGEPROTEIN
Therearesomestepsinthisprocess:-
Breakingofdisulfidebonds.
Disulfidebondsinterferewiththesequencing
procedure.Acysteinresiduethathasoneofits
peptidebondscleavedbytheEdmanprocedure
willremainattachedtoanotherpolypeptide.
Disulfidebondsalsointerferewiththe
enzymaticorchemicalcleavageofthe
polypeptide.
Twoapproachestoirreversiblebreakageof
disulfidebondsare–
Oxidationbyperformicacid.
Reductionbydithiothreitol.

19. Cleavingthepolypeptidechain
Proteiniscleavedintoasetofspecific
fragmentsbychemicalorenzymatic
methods.
Severalmethodscanbeusedfor
fragmentingthepolypeptidechain.Enzymes
calledproteasescatalyzethehydrolytic
cleavageofpeptidebonds.

21. Eachfragmentisthenpurifiedand
sequenced bytheEdman
procedure.
Finallytheorderinwhichthe
fragmentsappearintheoriginal
proteinisdetermined.

23. Massspectroscopy
Massspectrometersexplicitthedifference
inthemasstocharge(m/z)ratioofionized
atomsormoleculestoseparatethemfrom
eachothers.
The(m/z)ratioofmoleculesisalsoahighly
characteristicproperlythancanbeusedfor
determiningchemicaland structural
information.

24. Thebasicoperationofmassspectrometeris
Evaporationandionizemoleculesinvacuum,creating
agasphaseions.
Separatetheionsinspaceand/ortimebasedontheir
m/zratio.
Measuretheamountwithspecificm/zratio.

25. Theheatingorothertreatmentneededtotransfera
macromoleculetothegasphaseusuallycausedits
rapiddecomposition.Sodifferenttechniqueswere
developedtoovercomethisproblem.
i.Electrosprayionizationmassspectrometryor
ESIMS.
ii.Tandemmassspectrometry.

26. Macromoleculesinsolutionare
forceddirectlyfromtheliquidto
gasphase.
Asolutionofanalytesispassed
throughachargedneedlethatis
keptatahighelectricalpotential,
dispersingthesolutionintoafine
mistofchargedmicrodroplets.
Thesolventsurroundingthe
macromolecules rapidly
evaporates,andtheresulting
multiplychargedmacromolecular
ionsarethusintroduced
nondestructivelyintothegas
phase.Thistechniqueiscalled
electrosprayionizationmass
spectrometry,orESIMS.
Electrospray ionization mass spectrometry or ESI MS.

27. Tandem mass spectrometry
Asolutioncontainingtheproteinunder
investigationisfirsttreatedwithaproteaseor
chemicalreagenttohydrolyzeittoamixture
ofshorterpeptidesthenthemixtureis
injected.
Inthefirst,thepeptidemixtureissortedand
theionizedfragmentsaremanipulatedsothat
onlyoneoftheseveraltypesofpeptides
producedbycleavageemergesattheother
end.

28. Thentheselectedpeptidestravelthroughavacuumchamber
betweenthetwomassspectrometers.Inthiscollisioncell,the
peptideisfurtherfragmentedbyhigh-energyimpactwitha
“collisiongas,”asmallamountofanoblegassuchashelium
orargon.
Thesecondmassspectrometerthenmeasuresthem/zratios
ofallthechargedfragments.

30. APPLICATION OF PROTEIN
SEQUENCING
IdentificationofNewProteins.
ProbeDesignforMolecularCloning.
ManufactureofSyntheticPeptidesforUseas
Immunogens.
ProteinSequencesinEvolution.
Nowmanydatabasesareavailableonlinein
whichsequenceofproteinpresentwhichis
identified.

32. Introduction
Nucleicacidsarelargebiological
moleculesessentialforallknown
formsoflife.
They include DNA
(deoxyribonucleicacid)andRNA
(ribonucleicacid).Togetherwith
proteins,nucleicacidsarethemost
important biological
macromolecules;eachisfoundin
abundanceinalllivingthings,
wheretheyfunctioninencoding,
transmittingandexpressinggenetic
information.
Nucleicacidswerediscoveredby
FriedrichMiescherin1869.

33. Nucleic Acid Sequencing
Anucleicacidsequenceisasuccessionoflettersthat
indicatetheorderofnucleotideswithinaDNA(usingGACT)or
RNA(GACU)molecule.
Byconvention,sequencesareusuallypresentedfromthe5'
endtothe3'end.Becausenucleicacidsarenormallylinear
(unbranched)polymers,specifyingthesequenceisequivalent
todefiningthecovalentstructureoftheentiremolecule.
Thesequencehascapacitytorepresentinformation.
Sequencescanbereadfromthebiologicalrawmaterial
throughDNAsequencingmethods.

34. Type of Nucleic Acid Sequencing
DNA Sequencing
RNA Sequencing
There are two type of Nucleic Acid
Sequencing-

35. DNA Sequencing
DNAsequencingistheprocessofdeterminingthepreciseorderof
nucleotideswithinaDNAmolecule.
Itincludesanymethodortechnologythatisusedtodeterminethe
orderofthefourbases—adenine,guanine,cytosine,andthymineina
strandofDNA.
TheadventofrapidDNAsequencingmethodshasgreatlyaccelerated
biologicalandmedicalresearchanddiscovery.
KnowledgeofDNAsequenceshasbecomeindispensableforbasic
biologicalresearch,andinnumerousappliedfieldssuchasdiagnostic,
biotechnology,forensicbiology,andbiologicalsystematics.
DNAsequencingisoftentalkedaboutinthecontextoftheHuman
GenomeProject,inwhich,thecompletehumangenomewas
successfullysequencedin2001.

36. HISTORY
JamesWatsonandFrancisCrickpublishedthe
firstdescriptionofthecrystallographicdouble-
helixDNAstructurein1953.
In1977,Maxam andGilbertwasfirst
developed DNA sequencing technique by
usingchemicalreagents.
In1980,FredrickSangerdeveloped most
widelyusedmethodofDNAsequencingi.e.,
Chainterminationmethod.
TheSangerInstitutewasopenedin1993,
threeyearsaftertheinceptionoftheHuman
Genome Projectandplayedimportantrolein
sequencing the 8chromosome pairsof
human(1,6,9,10,13,20,22,andX).

37. Definition
“DNA Sequencing means finding the order
of nucleotides (adenine, guanine, cytosine,
and thymine) on a piece of DNA.”

38. METHODS OF DNA SEQUENCING
A)Maxam/Gilbertchemicalsequencing
DevelopedbyMaxamandGilbertin1977.
AstrandofsourceDNAislabeledatoneend
with
32
P.
ThetwostrandsofDNAaredistributedintofour
samples(inseparatetubes).
Eachsampleissubjectedtotreatmentwitha
chemicalthatspecificallydestroysone(G,C)or
twobases(A+G,T+C)intheDNA.

41. Advantages-
Rapid
Accurate
Limitations-
Thismethodiscomplex.
ItsometimesdamageDNAstrands
duringthetreatmentofchemicals.

42. Sanger Method
DevelopedbyFredrickSangerin1980.
AlsoknownasDideoxynucleotidemethodor
Chainterminationmethod.
4Steps:
1.Denaturation
2.Primerattachmentandextensionofbases
3.Termination
4.Gelelectrophoresis

44. Advantages-
BestforsmallDNAsegments.
Rapidandfast.
Limitations-
Theneedforasingle-strandedDNAtemplate.
Theuseofaprimertoanunknownsequence.
Thedideoxymethodisgoodonlyfor500-750bp
reactions.
ThismethodisExpensive.

45. Automated DNA Sequencing
Thehighdemandforlow-costsequencinghas
driventhedevelopmentofhigh-throughput
sequencing(ornext-generationsequencing)
technologiesthatparallelizethesequencing
process,producingthousandsormillionsof
sequencesatonce.

47. Advantages-
Itisdesirabletoacquiresequence
datainrealtimebydetectingtheDNA
bands withinthegelduringthe
electrophoreticseparation.
Itisarapidandaccuratetechnique.
Automated sequencercanaccurately
sequenceupto10,000nucleotidesper
day.
Thecostworksouttobenotmore
than$0.2pernucleotide.

48. DNA chips (Microarray Technique)
•DiscoveredbyShenaetalin1995.
•DNAsequencingasresultinadvancesmadeinautomation
andminiarization.
•AlargenumberofDNAprobes,eachonewithdifferent
sequence,areimmobilizedatdefinedpositionsonthesolid
surface.
•madeupofeithernylonorglass.
•TheprobescanbeshortDNAmoleculessuchascDNAsor
syntheticoligonucleotides.

50. Pyrosequencing
Visiblelightisgeneratedandisproportionaltothenumber
ofincorporatednucleotides
Determinewhichoneofthefourbases(A,G,C,T)is
incorporatedateachstepwhileaDNAtemplateiscopied.
withoutaddeddideoxynucleotides(ddNTPs)

51. Solid Phase Pyrosequencing
○Immobilized DNA
○3 enzymes
○Wash step to remove nucleotides after each addition

52. 3enzymes+apyrase
(nucleotidedegradation
enzyme)
Eliminatesneedfor
washingstep
Inthewellofamicrotiter
plate:
•primedDNAtemplate
•4enzymes
Nucleotidesareadded
stepwise.
Nucleotide-degrading
enzymedegradeprevious
nucleotides.
Liquid Phase Pyrosequencing

53. APPLICATION
Molecularbiotechnology
Forensicscience
GeneticEngineering
Clinicalapplication
Agriculture
HumanGenomeProject

54. INSTITUTES FOR DNA
SEQUENCING
A)InIndia
1.InstituteofGenomicsandIntegrativeBiology(CSIR-IGIB),Delhi
2.NationalBotanicalResearchInstitute(CSIR-NBRI),Lucknow
3.CentralDrugResearchInstitute(CSIR-CDRI),Lucknow
4.CentreforCellular&MolecularBiology(CSIR-CCMB),Hyderabad
5.NationalAidsResearchInstitute(NARI),Pune
B)InAbroad
1.TheWhiteheadInstitute/MITCenterforGenomeResearch,Cambridge.
2.GenomeSequencingCenter,Washington,UniversitySchoolofMedicine,
St.Louis.
3.TheWellcomeTrustSangerInstitute,Cambridge,England.
4.NationalCentreforGenomeSequence(Mexico).

55. Reference
Books
BiochemistrybyNelsonandCox,fifthedition,W.H.freemanandCompany
Newyork.
GeneCloningandDNAAnalysisbyT.A.Brown,sixthedition,AJohnWileyand
Sons,Ltd,publication,2010.
Internet
http://www.oswego.edu/~kadima/CHE525/PROTEIN%20SEQUENCING%20my%20l
ecture%20notes%20I.pdf
en.wikipedia.org/wiki/Protein
en.wikipedia.org/wiki/Nucelicacid
Journal-
MaxamA.M.andGilbertW.(1977)."AnewmethodforsequencingDNA".Proc.
Natl.Acad.Sci.U.S.A.74(2):560–4
Ronaghietal.(1996)Real-timeDNAsequencingusingdetectionofpyrophosphate
release.AnalyticalBiochemistry242(1):84–9
Ronaghietal.(1999)PyrosequencingShedsLightonDNASequencing.Genome
Res.2001.11:3-11