Exploring peptides in the brain by in vivo midrodialysis-capillary liquid chromatography-tandem mass spectrometry

154 Pages·2003·6.8 MB·English

by  Haskins, William E

EXPLORINGPEPTIDESINTHEBRAINBYINVIVOMICRODIALYSIS-
CAPILLARYLIQUIDCHROMATOGRAPHY-TANDEMMASSSPECTROMETRY
By
WILLIAME.HASKINS
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
AUGUST2003
ToLucasandKsenija.
ACKNOWLEDGMENTS
Myheartgoestothosewhogavemetheirloveandencouragementduringthese
difficultandwondrousyears. Itismyhonortothankmytwogreatestmentors,my
advisorDr.RobertT.KennedyandDr.DavidH.Powell;andmygreatestcolleague,Mr.
ChristopherJ.Watson,forhelpingmedosomethingIcanbeproudof,andforbeing
patientwithme. IalsothankthemembersoftheKennedyGroup,UFSpectroscopic
—
Services,UFChemistry;andmydoctoralcommittee Dr.BenHorenstein,Dr.Satya
—
Kalra,andDr.RichardA.Yost fortheirinsight. IthankAlfredChung(UFProtein
Core)forpeptidesynthesis;PeterViberg(LundUniversity)forhelpinemitter
production;WeihongTan(UFChemistry)foruseofalaserpipette-puller;andMaggie
Stolls,RogerReep,TomVickroy(UFPhysiologicalSciences)andJoannaPeris(UF
Pharmacology)forconfirmingmicrodialysisprobeplacement. IalsothankJames
Woods(UofMichiganPharmacology)forconductingopioidreceptorassays. Igratefully
acknowledgetheAnalyticalChemistryDivisionattheUniversityofFloridaforaLaitinen
Fellowship;theNationalInstitutesofHealth(NS38476);theNationalScience
Foundation;andBristol-MyersSquibbforfundingthiswork. Lastly,Iacknowledgemy
postdoctoralmentors,Dr.RonaldL.Hayes,Dr.KevinK.Wang,andDr.NancyD.
DenslowfortheirpatiencewithmewhileIwrotethisthesisandbeganexploring
traumaticbraininjuryattheMcKnightBrainInstitute.
iii
3
TABLEOFCONTENTS
Page
ACKNOWLEDGMENTS
iii
LISTOFFIGURES vi
LISTOFTABLES ix
LISTOFABBREVIATIONS x
ABSTRACT xii
CHAPTER
INTRODUCTION
1 1
SynpaticReleaseofNeuropeptides 1
InVivoMicrodialysis 3
CapillaryLiquidChromatography 5
ElectrosprayIonization 7
TandemMassSpectrometry 9
Multi-DimensionalTechniques 10
DissertationOverview 12
2 MONITORINGandDISCOVERINGENDOGENOUSNEUROPEPTIDESBYIN
VIVOMICRODIALYSIS-CLC-MS2 1
Introduction 13
Experimental 15
ResultsandDiscussion 19
Conclusion 40
3 DISCOVERINGENDOGENOUSNEUROPEPTIDESBYINVIVO
MICRODIALYSIS-CLC-DATA-DEPENDENT-MS2 41
Introduction 41
Experimental 43
ResultsandDiscussion 50
Conclusion 73
SupportingInformation 75
IV
1
4 STUDYINGENDOGENOUSNEUROPEPTIDEPROCESSINGBYINVIVO
MICRODIALYSIS-CLC-MS2 82
Introduction 82
Experimental 84
ResultsandDiscussion 84
Conclusion 105
5 SUMMARYANDFUTUREDIRECTIONS 106
Summary 106
FutureDirections 11
APPENDIX 115
A EXAMPLEOFCLC-MS2CALCULATIONS 115
B EXAMPLEOFPEPTIDESEQUENCINGANDPROTEINPRECURSOR
IDENTIFICATION 118
C TROUBLESHOOTINGTHEINVIVOMICRODIALYSIS-CLC-MS2
SYSTEM 124
LISTOFREFERENCES 125
BIOGRAPHICALSKETCH 140
v
1
LISTOFFIGURES
Figure Page
1-1. Synapticreleaseofneuropeptides 1
1-2. AmodelofproteolyticprocessingforPEA 3
1-3. Invivomicrodialysisscheme ...A
11--4. Photographofinvivomicrodialysisofafreelymovingrat 5
21--5. Scanningelectronmicrographofa25pm-i.dCLCcolumnpackedwith5pm
octadecylsilane(Cl8)particlesforpreconcentration,desaltingandseparationofattomole
amountsofpeptidesinmicrolitersamplevolumesbyreverse-phasegradient-elution 6
1-6. Photographofelectrosprayionization 8
1-7. Peptidesequencingbycollision-induceddissociation 9
8. Flowchartforourmulti-dimensionalmethod,invivomicrodialysis-CLC-MS,
developedformonitoringanddiscoveringpeptidesinthebrain 1
1. Blockdiagramoftheautomatedtwo-pressureCLC-MS2systemwithon-line
microdialysissampling 20
2-2. CLCcolumnswithintegratedelectrosprayemitters 22
2-3. Effectofgradientsteepnessparameter(b)onresolution(R)andsensitivity(peak
height)asafunctionofflowrate 24
2-4. Effectofgradientsteepnessparameter(b)onresolution(R)fordifferentcolumn
lengthsandflowrates(=2cmlongcolumnat20nL/min:•=10cmlongcolumnat50
nL/min;4=2cmlongcolumnat70nL/min) 25
2-5. HighsensitivityCLC-MS2measurementofamixtureofMet-enkephalinandLeu-
enkephalinat33pM(1.8(iLinjectedcorrespondingto59amolofeachpeptideloaded
ontothecolumn)eachinaCSFusinga2cmcolumn 27
vi
2-6. Illustrationofpeptidecarry-overintheinjectionvalveandcarry-overprevention,
usingdeuteratedstandards 29
2-7. InvivodetectionofMet-andLeu-enkephalinduringbasalandK+-stimulated
conditions 31
2-8. InvivomonitoringresultsforMet-enkephalin(),Leu-enkephalinO)andan
unknownpeptide( )forasinglerat 34
2-9. InvivoidentificationofpeptidesbyCLC-data-dependent-MS~ 36
2-
23--10. Sequencingofanovelpeptidebytime-segmentedMS fromsamplecollectedin
vivoduringK+-induceddepolarization 37
2-11. Confirmationofthesequenceofanovelpeptidebytime-segmentedMS from
samplecollectedinvivoduringK+-induceddepolarization .....38
12. AminoacidsequenceofPEA 39
1. Triplcate,0.4fiL,injectionsofatrypticdigestoflysozyme,carbonicanyhdrase,
andconalbuminat10nMwith1nMYGGFDsL(400amolinjectedon-column) 55
3-2. Invivomicrodialysis-CLC-MS2analysisofCSFcollectedfromtheratstriatum
duringK+-inducedreleaseconditions 56
3-3. Comparisonoftotalionchromatograms(TIC)andreconstructedion
chromatograms(RICs)fordata-dependentMS'ofdialysatecollectedfromratstriatum
duringbasal(BandD,respectively)anddepolarizing(AandC,respectively)conditions
showingthemostabundantprecursorionsobservedinvivo 57
3-4. RepresentativeMS2spectraandsequencesforpeptidesproducedfromproteolytic
processingofPEA(A),neurogranin(B),fibrinogena(C),fibrinogen(3(D),excitatory
aminoacidtransporter(E),andbrainacidicmembraneprotein(F) 63
3-5. MS2spectra(AandC)fromtheinvivolibraryandthedifferenceMS2spectrum
(B)forSPQLEDEAKEobservedintworats 66
3-6. Asummaryoftheinvivoresultsillustratingthesixstepdata-reductionstrategy
describedintheexperimentalsection 67
3-7. Diagramshowingtheinteractionofmultipleselectioncriteriaandtheoverlap
betweenMS"spectraremainingatdata-reductionsteps4and5(seeexperimental
section) 72
Vll
3-8. MS’spectraandsequencesforpeptidesproducedfromproteolyticprocessingof
PEA(A),neurogranin(B),fibrinogena(C),fibrinogen[3(D),excitatoryaminoacid
transporter(E)andbrainacidicmembraneprotein(F) 75
4-1. Invivomicrodialysis-CLC-MS2analysisofCSFcollectedfromtheratstriatum
duringdepolarization-inducedreleaseofpeptidesintotheECF 85
4-2. AminoacidsequenceofPEAandPEA-derivedpeptidesdiscoveredinthiswork93
44--3. Non-exhaustivedepictionofselectedPEA-derivedpeptidesbyaminoacid
p5o-sition.
99
4-4. Hypotheticalintermediates(His)ofPEAprocessingandnovelPEA-processing
observedinvivo 100
5. InvivoeffectofpeptideImoonneurotransmitterlevels 101
1. Invivomicrodialysis-CLC-MS2basepeakreconstructedionchromatograms
(RICs)showthemostabundantproductionsobservedduringsleeping(A)and
prolonged-wakefulness(B)states 113
B-l. Sequestsummaryreportforatrypticdigestofcytochromec 118
B-2. Sequestcyctochromecreportforatrypticdigestofcytochromec 119
B-3. Mascotsummaryreportforatrypticdigestofcytochromec 120
B-4. Mascotcytochromecreportforatrypticdigestofcytochromec 121
B-5. MascotannotatedMS2spectrumforthetrypticpeptideTGPNLHGLFGRfrom
thetrypticdigestofcytochromec 122
B-6. LutefiskreportforthetrypticpeptideTGPNLHGLFGRfromthetrypticdigestof
cytochromec 123
LISTOFTABLES
Table Page
33--1. Summaryofinvivomicrodialysis-CLC-MS2results 58
4-
3-2. Peptidesandproteolyticprocessingpatternsobservedinvivo 59
3-3. SummaryofinvitroCLC-MS2results 80
4. Peptidesandproteolyticprocessingpatternsobservedinvitro 81
1. Putativeproteasesinvolvedintheproteolyticprocessingpatternsobservedinvivo
88
A-1. ExampleofCLC-MS2calculations 115
C-l. Symptoms,diagnosisandtreatmentforcommonproblemsineachdimensionof
theinvivomicrodialysis-CLC-MS'system 124
IX
LISTOFABBREVIATIONS
aa AminoAcid
aCSF Artificialcerebralspinalfluid
amol Attomole
amu Atomicmassunits
BAM Bovineadrenalmedulla
BAMP Brain acidicmembraneprotein
CaM Calmodulin
CLC Capillaryliquidchromatography
EAAT1 Excitatoryaminoacidtransporter1
ECF Extracellularfluid
HI Hypotheticalintermediate
HPLC High-performanceliquidchromatography
id Internaldiameter
LC Liquidchromatography
pL Microliter
MS Massspectrometry
MS2 Tandemmassspectrometry
MWCO Molecularweightcut-off
NG Neurogranin
x