[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.

[application note]

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).

[application note]

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+).

[application note]

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

J.C. Silva, R. Denny, C.A. Dorschel, M.V. Gorenstein, I.J. Kass, G.-Z. Li, T. McKenna, M.J. Nold, K. Richardson, P. Young and S.J. Geromanos. Quantitative Proteomic Analysis by Accurate Mass Retention Time Pairs. Anal. Chem. 2005, 77, 2187-2200

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

J.C. Silva, R. Denny, C. Dorschel, M.V. Gorenstein, G.-Z. Li, K. Richardson, D. Wall, and S. J. Geromanos. Simultaneous Qualitative and Quantitative Analysis of the Escherichia coli Proteome: A Sweet Tale. Mol. Cell. Proteomics 2006, 5, 589 - 607

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