DTIC ADA506857: Identification of PVK/CAP2b Neuropeptides From Single Neurohemal Organs of the Stable Fly and Horn Fly via MALDI-TOF/TOF Tandem Mass Spectrometry

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peptides 27 (2006) 521–526
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Identification of PVK/CAP2b neuropeptides from single
neurohemal organs of the stable fly and horn fly via
MALDI-TOF/TOF tandem mass spectrometry
Ronald J. Nachmana,*, William K. Russellb, Geoffrey M. Coastc, David H. Russellb,
J. Allen Millerd, Reinhard Predela,e
aAreawidePestManagementResearch,SouthernPlainsAgriculturalResearchCenter,USDA,2881F/BRoad,
CollegeStation,TX77845,USA
bTheLaboratoryforBiologicalMassSpectrometry,DepartmentofChemistry,TexasA&MUniversity,CollegeStation,TX77843,USA
cSchoolofBiologicalandChemicalSciences,Birkbeck,UniversityofLondon,LondonWC1E7HX,UK
dKnipling-BushlandU.S.LivestockEntomologyResearchCenter,USDA,Kerrville,TX78028,USA
eSaxonAcademyofSciences,ResearchGroupJena,Erbertstrasse1,Jena07743,Germany
article info a bs t r ac t
Articlehistory: MALDI-TOF/TOFtandemmassspectrometryhasbeenappliedtodeterminethecomplete
Received14July2005 sequencesofthePVK/CAP2bneuropeptidesinthestableflyStomoxyscalcitransandhornfly
Accepted28July2005 Haematobiairritans,insectpestsoflivestock.Thispeptidomicanalysisofsingleneurohemal
Publishedonline2December2005 organpreparationsallowstheunambiguousassignmentofinternalLeu/Ilepositionsnot
distinguishablebypreviousmassspectrometrictechniques.Thesequencesareasfollows:
Keywords: Stoca-PVK/CAP2b-1, AGGASGLYAFPRVa; Stoca-PVK/CAP2b-2, NAKLYPVPRVa; and Haeir-
MALDI-TOF/TOFmassspectrometry PVK/CAP2b-1, AGGASGLYAFPRVa; Haeir-PVK/CAP2b-1, NAKLYPMPRVa. Both Stoca-PVK/
Insect CAP2b-1 and -2 stimulate Malpighian tubule fluid secretion in the stable fly, with EC50
Neuropeptide valuesbetween3and11nM.Theidentificationofthesenovelneuropeptidesaddstoour
Periviscerokinin knowledgeofthepeptidomesofflies,andcanaidinthedevelopmentofneuropeptide-based
CAP2b controlstrategiesoftheseinsectpests.
Stomoxyscalcitrans PublishedbyElsevierInc.
Haematobiairritans
Peptidomics
1. Introduction viadirectanalysisofsingleneurosecretoryorgansornerves,
includingthoseofinsects[13,18,19]viathepost-sourcedecay
Neuropeptidesareimportantmessengermoleculesthatoccur (PSD) technique. Alternatively, electrospray ionization (ESI)
inagreatvarietyofformsandareimplicatedintheregulation coupledwithtandemMShasresultedintheidentificationofa
ofcriticalphysiologicalprocessessuchasdiuresis,digestion, numberofnovelneuropeptides[2,20,22,24,25].Theamountof
development and reproduction [6,7] in insects. In the past materialneededforESI-MSexperimentsisusuallylargerthan
several years, new developments in matrix-assisted laser theamountnecessaryforMALDI-MS,sincethepeptideshave
desorption-time-of-flightmassspectrometry(MALDI-TOFMS) to be extracted from the tissue prior to analysis. Both
haveaffordedverysensitivedenovosequencingofpeptides techniques, however, alleviate the need for large numbers
* Correspondingauthor.Tel.:[phone];fax:[phone].
E-mailaddress:[email protected](R.J.Nachman).
0196-9781/$–seefrontmatter.PublishedbyElsevierInc.
doi:10.1016/j.peptides.2005.07.022
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1. REPORT DATE 3. DATES COVERED
JUL 2005 2. REPORT TYPE 00-00-2005 to 00-00-2005
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Identification of PVK/CAP2b neuropeptides from single neurohemal
5b. GRANT NUMBER
organs of the stable fly and horn fly via MALDI-TOF/TOF tandem mass
spectrometry 5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
U.S. Department of Agriculture,Areawide Pest Management REPORT NUMBER
Research,2881 F/B Road,College Station,TX,77845
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522 peptides 27 (2006) 521–526
ofspecimensandthetime-consumingandexpensiveefforts on the ABI 4700 proteomics analyzer (Applied Biosystems,
required to isolate and determine the primary structure of Framingham,MA) [12]. Due to the nature of the samples all
neuropeptidesviatraditionalchromatographicandchemical acquisitions were taken in manual mode. Initially the
sequencing techniques [4,8,28]. These MS techniques have instrument was operated in reflectron mode, in order to
also made it possible to make detailed comparisons of the determinetheparentmasses.Thelaserintensitywassetjust
peptide patterns or profiles (i.e., the peptidomes) of closely abovethethresholdrequiredtoionizetheneuropeptides.For
relatedinsectspecies[1,3,24].MSanalysisofinsectneuropep- thetandemMSexperiments,theaccelerationvoltageapplied
tideshasfailedinthepasttodistinguishbetweentheisomers was1kVinallcases,andthelaserintensitywasincreasedby
LeuandIle,whichhaveidenticalmasses.Theseearlierstudies 10%. The number of laser shots used to obtain a spectrum
were limited to low energy fragmentations of the ion of variedfrom500to5000,dependingonsignalquality.Inorder
interest. A primary limitation of PSD peptide sequencing is tochangethenetamountofcollisionstotheprimaryionsin
thattheinternalenergiesofthe[M+H]+ionsarenotsufficient the collision induced dissociation (CID) experiment, the
toyieldthesidechaincleavagesnecessarytodistinguishLeu collision cell gas (atmospheric air) pressure was increased.
andIle.However,recentinnovationsinMALDI-TOFMShave Allthreegaspressuressettingsavailable(none,mediumand
allowedanalysisofhigh-energycollision-induceddissociation high)wereemployed.Theinstrumentwasoperatedinpost-
oftheparentionsofpeptidesthatrevealuniquemasspatterns source decay mode when no collision gas is used. The true
for the sidechains of Leu and Ile [11,12]. Indeed, in a recent pressure within the collision cell cannot be measured. The
study we have demonstrated the utility of MALDI-TOF/TOF fragmentation patterns from these three different settings
tandem MS in distinguishing between Leu and Ile in were used to determine the sequence of the peptide. The
neuropeptides from single neurohemal preparations of fragmentation data obtained in these experiments was
insects,specificallyPVK/CAP2b (periviscerokinin/cardioacce- handled using the Applied Biosystems Data Explorer1 soft-
leratory peptide 2b) sequences from the housefly (Musca warepackage.
domestica)andfleshfly(Neobellieriabullata)[15].
In this study, we utilize MALDI-TOF/TOF tandem MS to 2.3. Peptidesynthesis
undertake the first identification of the sequences of PVK/
CAP2bneuropeptides,includingtheunambiguousassignment Stoca-PVK-1/Haeir-PVK-1 (AGGASGLYAFPRVa), Stoca-PVK-2
of Leu/Ile positions, from single neurohemal organ prepara- (NAKLYPVPRVa), and Haeir-PVK-2 (NAKLYPMPRVa) were
tionsofadultsofthestableflyStomoxyscalcitransandhornfly synthesized via Fmoc methodology on Rink Amide resin
Haematobiairritans,importantlivestockpests.PVK/CAP2bsare (Novabiochem, San Diego, CA) using Fmoc protected amino
typicaloftheabdominal neurohemal systemofinsects[29], acids (Advanced Chemtech, Louisville, KY) on an ABI 433A
usuallystoredinabdominalperisympatheticorgans.ThePVK/ peptidesynthesizer(AppliedBiosystems,FosterCity,CA)under
CAP2b class of neuropeptides has beenshown to elicit both previously described conditions [14]. Crude products were
myotropicactivityandstimulationofMalpighiantubulefluid purifiedonaWatersC SepPakcartridgeandaDeltaPakC
18 18
secretionininsects(see[29]),physiologicalprocessescritical reverse-phasecolumn(8mm(cid:1)100mm,15(mparticlesize,100
tosurvival.WereportonthediureticactivityofthetwoPVK/ (poresize)onaWaters510HPLCcontrolledwithaMillennium
CAP2b sequences native to the stable fly in a stable fly 2010chromatographymanager system(Waters,Milford,MA)
Malpighiantubulefluidsecretionbioassay. with detection at 214nm at ambient temperature. Solvent
A=0.1% aqueous trifluoroacetic acid (TFA); Solvent B=80%
aqueous acetonitrile containing 0.1% TFA. Conditions: Initial
2. Materials and methods solventconsistingof20%BwasfollowedbytheWaterslinear
programto100%Bover40min;flowrate,2ml/min.Delta-Pak
2.1. Insects C-18 retention times: Stoca-PVK-1/Haeir-PVK-1 (AGGAS-
GLYAFPRVa),9.0min;Stoca-PVK-2(NAKLYPVPRVa),6.25min;
Stable flies (S. calcitrans) and horn flies (H. irritans) were and Haeir-PVK-2 (NAKLYPMPRVa), 10.95min. The peptides
obtainedaspupaefromtheKnipling-BushlandU.S.Livestock were further purified on a Waters Protein Pak I125 column
InsectsLaboratoryinKerrville,TX.Pupaewerekeptincages (7.8mm(cid:1)300mm) (Milligen Corp., Milford, MA). Conditions:
at268C.Newlyemergedstableandhornflieswerefedabeef Flowrate:2.0ml/min;isocraticwithsolvent=80%acetonitrile
blood meal twice daily for two weeks and provided with madeto0.01%TFA.WatProretentiontimes:Stoca-PVK-1/Haeir-
sucroseandwateradlibitum.Maleandfemaleflies<10days PVK-1 (AGGASGLYAFPRVa), 9.25min; Stoca-PVK-2 (NAK-
post-emergence were used in all experiments. Stable flies LYPVPRVa), 8.75min; and Haeir-PVK-2 (NAKLYPMPRVa),
usedinfluidsecretionassayswereobtainedaspupaefrom 9.0min.Aminoacidanalysiswascarriedoutunderpreviously
Professor Mike Lehane (Liverpool School of Tropical Medi- reportedconditions[14]andusedtoquantifythepeptideandto
cine).Adultflieswereheldat278Candfeda5%solutionof confirmidentity,leadingtothefollowinganalyses:Stoca-PVK-
sucrose. 1/Haeir-PVK-1(AGGASGLYAFPRVa):A[2.8],F[1.0],G[2.7],L[1.0],
P[1.1],R[1.0],S[0.9],V[1.1],Y[1.0];Stoca-PVK-2(NAKLYPVPRVa),
2.2. Matrix-assistedlaserdesorptionionizationtandem A[1.0],K[0.9],L[1.0],N[0.9],P[1.9],R[1.0],V[1.9],Y[1.0];andHaeir-
time-of-flightmassspectrometry(MALDI-TOF/TOFMS) PVK-2(NAKLYPMPRVa),A[1.0],K[1.0],L[1.0],M[1.0],N[1.0],P[2.0],
R[1.0], V[1.0], Y[0.9]. The identity of the peptide analogs was
Dissection and sample preparation were performed as confirmed via MALDI-TOF-MS on a Kratos Kompact Probe
previously described [14,18]. MALDI analysis was performed MALDI-TOFMSmachine(KratosAnalytical,Ltd.,Manchester,
peptides 27 (2006) 521–526 523
UK)withthepresenceofthefollowingmolecularions(M+H+):
Stoca-PVK-1/Haeir-PVK-1(AGGASGLYAFPRVa),1266.3[M+H+];
Stoca-PVK-2(NAKLYPVPRVa),1156.6[M+H+];andHaeir-PVK-2
(NAKLYPMPRVa),1188.9[M+H+].
2.4. IsolatedstableflyMalpighiantubulepreparations
FluidsecretionfromisolatedstableflyMalpighiantubuleswas
measuredusingthe‘‘Ramsayassay’’aspreviouslydescribed
forhouseflytubules[8].Tubuleswereremovedfrom4-to7-
day-old adult flies of both sexes. Flies were dissected under
Muscasaline[8]andbothanteriorandposteriortubuleswere
transferredtosmall(10ml)dropsofbathingfluid(a1:1mixture
ofsalineandSchneider’sDrosophilamedium)beneathwater-
saturatedliquidparaffininaSylgardTMlinedPetridish.The
tubules were allowed to equilibrate for 1h before being
challengedwithtestpeptides.Urineescapedfromthecutend
of the tubule, which was pulled out into the liquid paraffin.
Drops of urine were collected at 15min intervals and their
diameter(d)measuredastheyrestedontheSylgardbaseof
the Petri dish using a Wild digital (MMS235) eyepiece
micrometer. Urine volume was calculated as pd3/6 and the
rateofsecretionobtainedbydividingthesecretedvolumeby
thecollectionperiod.Datawerenormalizedbyexpressingthe
increaseinfluidsecretionasapercentageoftheresponseto
Fig.1–MALDI-TOFmassspectraofpreparationsofthe
10nMMusdo-K(alsonativetothestablefly[13]),whichwas
posteriorabdominaldorsalsheathoftheventralnerve
added to all tubules at the end of each experiment. Dose–
cordofthestableflyS.calcitrans(A)andthehornflyH.
responsecurveswerepreparedusingthecomputerprogram
irritans(B).Thedorsalsheathrepresentsaneurohemal
GraphPadPrismversion4.02(GraphPadSoftware,SanDiego,
releasesitehomologoustoperisympatheticorganswhich
CA).
becomeincorporatedintothedorsalganglionicsheath
duringthemetamorphosisofcycloraphousflies.TheMH+
valuesareasfollows:forStoca-PVK/CAP2b-1(1264.70),
3. Results
Stoca-PVK/CAP2b-2(1155.72),Heair-PVK/CAP2b-1
(1264.68),andHeair-PVK/CAP2b-2(1187.67).
3.1. DeterminationofPVK/CAP2bsequencesin
abdominaldorsalsheathsoffliesviaMALDI-TOF/TOFmass
spectrometry
Direct analysisof abdominal dorsal sheath tissues from the
stableflyS.calcitransandhornflyH.irritanswereconductedvia
MALDI-TOF/TOFMS.IllustratedinFig.1aretheinitialMALDI-
TOFspectraofthepreparationsofthesetwofliesthatfeature
theparentionsofthePVK/CAP2bpeptides.HighenergyCIDof
the PVK/CAP2b peptides reveal unique patterns for the
sidechains of Leu and Ile [11,12,15]. As illustrated in Fig. 2,
fragments of native Stoca-PVK-2 of the stable fly include a
prominent ‘w7a’ fragment ion at mass 788.3Da (Fig. 2),
indicative of Leu in this peptide. Indeed, the spectrum of
the synthetic version of Stoca-PVK-2 containing Leu at
position 7 taken under the same conditions was essentially
identical.Incontrast,ifStoca-PVK-2containedIle,themass
spectrumunderconditionsofhighgaswouldrevealadifferent
mass for the ‘w7a’ fragment, along with two diagnostic
satelliteions,a‘v-ion’anda‘wb-ion’.Thus,Stoca-PVK-2can
be unambiguously assigned the sequence NAKLYPVPRVa
(Table 1). Using the same single organ preparations, the Fig.2–AMALDI-TOF/TOFtandemmassspectrumofnative
sequences of Stoca-PVK-1, Haeir-PVK-1, and Haeir-PVK-2 Stoca-PVK-2underconditionsofhighgaspressure(arrow
could also be unambiguously determined under conditions identifiesthe‘w7a’fragmentionat788.3).Themass
of high gas pressure to be AGGTSGLYAFPRVa, AGGTS- spectraofthenativeandsyntheticLeuvariantare
GLYAFPRVa,andNAKLYPMPRVa(Table1),respectively. essentiallyidentical.
524 peptides 27 (2006) 521–526
Table1–AminoacidsequencesofPVK/CAP2bpeptides Table2–FluidsecretionactivityofnativeStomoxys-
nativetothestablefly(S.calcitrans)andhornfly(H. PVK/CAP2b-1and-2onstablefly(S.calcitrans)Malpigh-
irritans)determinedbyMALDI-TOF-TOFtandemmass iantubules
spectrometrycomparedwithpreviouslydetermined Peptide Sequence Fluidsecretiona
sequencesfromotherflies
[EC ](nM)
50
Species PVK/CAP2b-1 PVK/CAP2b-2
Stoca-PVK/CAP2b-1 AGGASGLYAFPRVa 3.4(0.6–18.9)
S.calcitrans AGGASGLYAFPRVa NAKLYPVPRVa Stoca-PVK/CAP2b-2 NAKLYPVPRVa 11.0(3.8–31.6)
H.irritans AGGASGLYAFPRVa NAKLYPMPRVa
a Valuesinparentheses,representthe95%confidencelimit(CL).
M.domestica AGGTSGLYAFPRVa ASLFNAPRVa[15]
N.bullata NGGTSGLFAFPRVa AGLIVYPR[L/I]aa[15]
D.melanogaster GANMGLYAFPRVa ASGLVAFPRVa[27]
abdominal ganglia and are accumulated in perisympathetic
An.gambiae GPTVGLFAFPRVa QGLVPFPRVa[17]
organsuntilrelease.Larvalperisympatheticorgansofcyclor-
a MALDI-TOF/TOF tandem MS cannot distinguish between Leu aphousDiptera,however,becomeincorporatedintothedorsal
andIleataC-terminalposition[14]. ganglionic sheath [16] during the metamorphosis. The
abdominaldorsalsheath,whichwasdissectedinthisstudy,
therefore contains relatively large amounts of peptidergic
3.2. FluidsecretionactivityofPVK/CAP2bsequenceson neurohormones. The molecular ions and incomplete
stableflyMalpighiantubules sequences observed in the MALDI-TOF/TOF mass spectro-
metricstudiesoperatedinreflectronmode(Fig.1)onthestable
SyntheticreplicatesofthetwoPVK/CAP2bsequencesnativeto flyandhornflywere,Stoca-PVK-1(AGGASG[L/I]YAFPRVa;m/z
thestableflywereevaluatedfordiureticactivityonisolated 1264.70)andStoca-PVK-2(NAK[L/I]YPVPRVa;m/z1155.72);and
Malpighiantubules.AscanbeseeninFig.3andTable2,Stoca- Haeir-PVK-1 (AGGASG[L/I]YAFPRVa; m/z 1264.68) and Haeir-
PVK-1 and Stoca-PVK-2 stimulated stable fly Malpighian PVK-2 (NAK[L/I]YPMPRVa; m/z 1187.67). When the peptides
tubule fluid secretion with observed EC values of 3.4 and were fragmented under conditions of high collision energy,
50
11.0nM, respectively (Table 2). The peak response to both the collision-induced fragments reveal distinct side-chain
peptideswas(cid:2)25%ofthatobtainedaftertheadditionof10nM fragmentations. Stoca-PVK-2 demonstrated a fragmentation
Musdo-K(alsonativetothestablefly[13]),andrepresentedan patternindicativeofaLeuinposition7(Fig.2),andthissame
80%increaseoverthebasalrateoffluidsecretion. fragmentation pattern was observed in mass spectra of a
syntheticreplicatecontainingLeu.Thus,Stoca-PVK-1canbe
unambiguously assigned the sequence NAKLYPVPRVa
4. Discussion (Table1).Usingthesingleorganpreparations,thesequences
of Stoca-PVK-1, Haeir-PVK-1 and Haeir-PVK-2 could also be
PVK/CAP2b peptides have been found in the stable fly S. unambiguouslydeterminedunderconditionsofhighcollision
calcitransandhornflyH.irritansviaMALDI-TOFMSanalysisof energy to be AGGASGLYAFPRVa, AGGASGLYAFPRVa and
preparationsofneurohemaltissues[24].PVK/CAP2bpeptides NAKLYPMPRVa (Table 1), respectively. Thus, the PVK/
of insects are typical of neurosecretory neurons in the CAP2b-1sequencesofthestableflyandhornflyareidentical,
andthePVK/CAP2b-2sequencesdifferonlybyasingleresidue
inthefourthpositionfromtheC-terminus(VversusM).
ItisclearthattheLeuatthepositionlocatedsevenresidues
fromtheC-terminusinthesefourPVK/CAP2bsequencesfrom
stableflyandhornflyisconservedwithinandacrossspecies
(Table1).LeuatthisspecificpositionistypicalofotherPVK/
CAP2bofinsectsthatweresequencedbyEdmandegradation
inearlierstudies[5,9,20,21,23]orforwhichgeneshavebeen
publishedalready[10,26,27].Ingeneral,PVK/CAP2bsequences
arewellconservedthroughoutthesixspeciesoffliesthathave
beenstudiedtodate(Table1).
BoththestableflypeptidesstimulatedMalpighiantubule
fluid secretion, with Stoca-PVK/CAP2b-1 being slightly more
potentthanStoca-PVK/CAP2b-2,althoughthedifferencewas
notsignificant.Themaximumresponsewasequivalenttoan
80%increaseinthebasalrateofsecretion,whichisconsistent
withdataforotherdipteraninsects[17],andisabout25%of
theresponseofstableflytubulesto10nMMusdo-K.
Insummary,MALDI-TOF/TOFtandemMShasbeenusedto
identify the sequences for the PVK/CAP2b neuropeptides in
thestableflyandhornflyviadirectanalysisofnervetissue.
Fig.3–Dose–responsecurvesforStoca-PVK/CAP2b-1(solid This analysis includes an unambiguous assignment of the
lineandsymbols)and-2(dashedlineandopensymbols). monoisotopic residuesLeu versus Ile. The work adds to our
DataareexpressedasthemeanWS.E.Mforsixreplicates. knowledgeofthepeptidomesofflies;andtheidentificationof
peptides 27 (2006) 521–526 525
the specific structures of the PVK/CAP2b neuropeptides, dissociationusingmatrix-assistedlaserdesorption/
implicated in the regulation of diuretic and myotropic ionizationtandemtime-of-flight(MALDI-TOF/TOF)for
proteinandpeptideidentification.RapidCommunMass
processes, may aid in the development of mimetic analogs
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This study was supported by a Binational Agricultural
flyandhornfly-massspectrometricidentificationfrom
Research and Development Grant (BARD #IS-3356-02) (RJN),
singlenervesanddiureticactivity.Peptides2002;23:1885–
agrantfromDeutscheForschungsgemeinschaft(Predel595/6- 94.
1),aCollaborativeResearchGrant(#LST.CLG.979226)fromthe [14] NachmanRJ,IsaacRE,CoastGM,HolmanGM.Aib-
NorthAtlanticTreatyOrganization(NATO)(RJN,GMC),agrant containinganaloguesoftheinsectkininneuropeptide
from the USDA/DOD DWFP Initiative (#0500-32000-001-01R) familydemonstrateresistancetoaninsectangiotensin-
convertingenzymeandpotentdiureticactivity.Peptides
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