Identification and Quantification of Diagnostics Markers ...[application note] IdentIfIcatIon and...
Transcript of Identification and Quantification of Diagnostics Markers ...[application note] IdentIfIcatIon and...
[application note]
Ident If Icat Ion and Quant If Icat Ion of dIagnost Ics Mark ers and Pat hway analysIs for gaucher dIsease by Means of lc /Ms
Hans Vissers1, Jim Langridge1, Hans Aerts2 1 Waters Corporation, MS Technologies Centre, Manchester, UK 2 Academic Medical Center, University of Amsterdam, Department of Biochemistry, Amsterdam, T he Netherlands
oV erV Iew• Quantitative label-free nanoscale LC/MSE analysis has been
applied to the serum analysis of Gaucher disease patients to
identifyandquantifydiseasemarkersandindicators.
• Absolute and relative LC/MSE quantification analysis were
utilizedtoassesstreatmenteffectsandenzymeactivities.
• Clusteringapproacheswillbepresentedatthepeptide(accurate
mass-retention time cluster) and protein level to assess data
qualityandtreatmenteffects,respectively.
• Theuseofpeptide intensityprofilingwillbedemonstrated to
conductpathwayanalysis.
Int roduct Ion
Themostfrequentlyencounteredinherentlysosomalstoragedisorder
isGaucherdisease,whichismarkedbydeficiencyinglucocerebro-
sidase activity that catabolizes glycosylceramide to ceramide and
glucose. The latter leads to problems in biomolecular component
recycling.Theclinicalpresentationisheterogeneouswithrespectto
age,nature,andsymptomprogression.
Manifestationsareusuallyaccompaniedbyabnormalities inserum
composition. These are normally not attractivemarker candidates
since their prevalence varies. Patients are treated using enzyme
supplementationtherapytoalleviatesymptoms,Figure1.Recently,
anenzymehasbeendiscovered that iselevated inserumand isa
sensitive indicatorofGaucherdiseaseanda tool tomonitor treat-
mentefficacy.
PatientdigestserumsampleswereanalyzedbymeansofLC/MS.The
tandemmassspectrometeracquireddatainaspecialmodeofacqui-
sitionwherethecollisionenergywithinthecollisionenergywithin
Figure 1. Effect of two-year of enzyme replacement therapy (spleen and liver functionality restored).
thegascell iscontinuouslyswitched fromlowtoelevatedenergy,
withnoprecursorselectionbythefirstmassanalyzer.
Absolute protein concentration determination results for
Chitotriosidase–aspecificbiomarkerofGaucherdiseaseforsymp-
tomatic type I patients – will be presented. The effect of enzyme
replacementtherapywasmonitoredbylookingattheserumprotein
compositionasawhole.Bothundepletedanddepletedquantitative
LC/MSserumanalyseswereutilized for the latteras theycomple-
ment each other in terms of addressing the dynamic concentration
rangewithinserum.
Variousclusteringapproacheswillbedemonstratedtocharacterize
theinvestigatedstagesoftreatment.Proteinlevelclusteringwillbe
demonstrated toassess condition similarityand treatmenteffects.
Peptide intensityprofiling–withassociatedprotein identifications
–willbeusedtoidentifypathwayassociatedproteins.
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eX PerIMental
sample preparation
Patient serum samples – control, pre- and post-treatment – were
diluted prior to depletion with a 10 cm x 4.6 mm multi-affinity
removalsystemcolumnwhichremovesthesixmostabundantserum
proteins. Serum samples – both depleted and undepleted – were
subsequentlydenaturedwithRapiGest™SFsurfactant(0.1%)reduced
(10mMDTT), alkylated (10mM IAA),andenzymaticallydigested
withtrypsin,1:50(w/w)enzyme/proteinratio.
RapiGestwasremovedbytheadditionof2µLconc.HCl–followedby
centrifugation–andthesupernatantcollected.Sampleswerediluted
with0.1%formicacidtoanappropriatefinalworkingconcentration
prior to analysis – corresponding to an 0.5 µg of protein digest
on-column.
lc conditions
LC/MS quantification experiments were conducted using a 1.5 hr
reversed-phasegradientat250nL/min(5to40%acetonitrileover
90min)ontheWaters® IdentityEHighDefinitionProteomicsTM System,
usingasaninletthenanoACQUITYUPLC® System and an Atlantis®
3µmC18 NanoEase™75µmx15cmnanoscaleLCcolumn.Samples
wererunintriplicate.
Ms conditions
The IdentityE System also included the Q-Tof Premier™ Mass
Spectrometer,whichwasprogrammedtostepbetweennormal(5eV)
andelevated(15to35eV)collisionenergiesonthegascell,usinga
scantimeof1.5sperfunctionoverm/z50to1990.
data processing and protein identification
Proteinidentifications,dataalignment,andquantitativeinformation
were generated by the use of dedicated algorithms, including the
comprehensivepeptideionaccountinginformaticsincludedwiththe
IdentityESystem,andbysearchingvarioushumanspecificdatabases,
Figure2.Additionalstatisticalandclusteringdataanalysis,basedon
thecomma-separatedvaluesoutputfromtheExpressionE informatics
software,wasperformedwithDecisionsite(Spotfire),Excel(Microsoft)
andSimca-P+(Umetrics).
Figure 2. The IdentityE System search results browser embedded in ProteinLynx Global SERVER™ 2.3 Software.
[application note]
results
relative quantification
The low-energy data are used for quantification of the proteins,
whereasthehigh-energyinformationisutilizedforqualitativeiden-
tificationpurposes.Thetotalnumberofidentifiedproteinswas108.
46proteinswereidentifiedincommon,20werefoundtobeunique
totheundepletedserum,and42proteinswereuniquelyidentifiedin
thedepletedserumsamples.Approximately50percentofthepro-
teinsuniquelyidentifiedtobothdepletedandundepletedserumwere
foundtoberegulated.Figure4illustratestherelativequantification
resultfordepletedserum.
absolute quantification
Chitotriosidase is a known biomarker for symptomatic Gaucher
disease patients andwas positively identified in all pre-treatment
serumsamples.Theabsoluteconcentrationandenzymeactivityof
Chitotriosidase was determined by means of a recently published
absoluteconcentrationformula:
TheabsoluteconcentrationofdetectedChitotriosidasewasfoundto
beequalto1.6fmol/µLinthepresenceof0.5µgofdepletedserum,
corresponding to an estimated enzyme activity of 39,500 nmol/
mL.h.Theactivitymeasuredbymeansof theenzymaticassaywas
31,800nmol/mL.h.
data clustering – multivariate analysis
Theresultsoftheuseofprincipalcomponentanalysis(PCA)atthe
proteinlevelareshowninFigure4.Theproteinidentificationresults
forthedepletedandundepletedserumsampleswereannotatedwith
absolute determined amounts injected on-column. However, PCA
canbeskewedby thepresenceofuniqueproteins in thecaseofa
comparison between the depleted and undepleted serum samples.
Therefore,onlythecross-sectionoftheproteinsthatwereidentified
inallinjections–withinallconditions–wasusedforPLS-DA.From
theresultsshowninFigure4,thefirsttwomainprincipalcomponents
canbeeasilyidentifiedastreatmentanddepletion,respectively.
[ ]standard internalstandard internalintensity peptide normalized
xproteinintensity peptide normalized
n
1i
n
1i ⋅∑
∑
=
=
Figure 3. Relative protein concentration (elog ratio) of the pre- and post-treatment depleted serum samples versus depleted control serum (grey bars = pre-treatment; red bars = post-treatment).
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Figure5illustratestheclusteringofthepeptideintensitiesbytheir
pattern, allowing protein identification of proteins involved in the
samebiologicalprocess–hence,allowingpathwayanalysis.
Forexample,theleftpanel(a)showsaclusterthatwaslowininten-
sitypre-treatmentandincreasedinintensitypost-treatmentandalso
inthecontrolsample.Thethreeleftcolumnscorrespondtothetrip-
licateinjectionsofpost-treatment,themiddlethreetopre-treatment
andtherightthreetocontrolserumsamples.Theprofiledpeptides
can bemapped to their parent proteins. For example, 15 proteins
contributedwithat least threepeptidesto theprofilecluster (a) in
Figure5,ofwhich sixarepartof the complementandcoagulation
cascades.Asimilar lower intensityprofile identifiedanadditional
eightproteinsthatarepartofthesamepathway.Hence,allproteins
identified in the complement pathway show the same regulation
trends,asshowninFigure6.
TheprofileclusterinFigure5(b)representsproteinsthatareabundant
inthepre-andpost-treatmentsampleandarenotidentifiedinthe
control.ThesecorrespondtoFibrinogenalphachain,Fibrinogenbeta
chain,andFibrinogengammachain.TheserumofGaucherpatients
istypicallycharacterizedbysevereabnormalitiesinthecoagulation
system,contributing to the tendencyofGaucherpatients tobleed.
Thisiscompensatedbythefibrinolyticsystem.
ThethirdclusterrepresentspeptidesfromApolipoproteinA1,aprotein
down-regulated pre-treatment that is onlymarginally normalizing
upontherapy.ApolipoproteinA1isacomponentoftheHDLserum
content,whichisextremelylowandknowntobepoorlynormalized
followingenzymereplacementtherapy.
In addition to the presented clustering approaches, cluster-driven
geneontologysearchescanbeperformedtodeterminethecorrela-
tionbetweenthemolecularfunctionsoftheidentifiedpeptides,and
potentially,theassociatedproteins.Forinstance,fortheclustershown
inFigure6(a),themolecularfunctiondistributionbasedonretrieved
geneswas42%binding,11%catalyticactivity,25%enzymeregula-
toractivity,11%signaltransduceractivity,11%transporteractivity
byannotatingtheclusterwithgeneindexes.
Figure 4. PLS-DA – Non-centered, Pareto-scaled – of both depleted and undepleted samples utilizing the measured absolute serum protein concentrations (Simca-P+).
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Figure 5. Intensity profile (K-means) clustering examples of peptides identified to a protein for the depleted serum samples – post-treatment (left columns), pre-treatment (middle columns) and control serum (right columns).
Figure 6. Complement cascade with the identified complement proteins in color and absolute determined concentrations – pre-treatment (blue), post-treatment (green) and control (black).
[application note]
waters corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
conclusIons• Enzymereplacementtreatmentefficacyhasbeendemonstrated
bymeansofuni-andmulti-varianceLC/MSanalysisofdepleted
andundepletedserum.
• A specific Gaucher disease biomarker – Chitotriosidase – has
beenpositively identified,quantified,and itsenzymeactivity
determined.
• Principalcomponentanalysis(PCA)canbeappliedtoboththe
lowandelevatedenergydatasetstorapidlyassessdataquality
andidentifytrends.
• Clusteringgeneontology-drivensearchesprovidesaconvenient
meansforclassificationofthepeptidesandproteinsthatexhibit
a similar profile change from a complex data set to identify
pathwayassociatedproteins.
• Theresultsobtainedwiththepresentedlabel-freequantitative
MSmethodsareconsistentwithbiochemicalfindings.
references
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J.C. Silva, M.V. Gorenstein, G.-Z. Li, J.P.C. Vissers and S.J. Geromanos. Absolute Quantification of Proteins by LC-MSE; a Virtue of Parallel MS Acquisition. Mol. Cell. Proteomics 2006, 5, 144-156
M.A. Huges, J.C. Silva, S.J. Geromanos and C.G. Townsend. Qunatitaitve Proteomic Analysis of Drug-Induced Changes in Mycobacteris. J. Proteome Res 2006, 5, 54-63
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