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Sugarnucleotidequantificationbyliquidchromatographytandemmassspectrometryrevealsa
distinctprofileinPlasmodiumfalciparumsexualstageparasites
BorjaLópezͲGutiérrez1,RhoelR.Dinglasan2andLuisIzquierdo1*
[1]ISGlobal,BarcelonaCtr.Int.HealthRes.(CRESIB),HospitalClínic–UniversitatdeBarcelona,
Barcelona,Spain
[2]TheUniversityofFloridaEmergingPathogensInstitute,DepartmentofInfectiousDiseases
&Pathology,Gainesville,Florida32611,USA
*Towhomcorrespondenceshouldbeaddressed([email protected])
1
Abstract
The obligate intracellular lifestyle of Plasmodium falciparum and the difficulties to obtain
sufficient amounts of biological material has hampered the study of specific metabolic
pathwaysinthemalariaparasite.Thus,forexample,thepoolsofsugarnucleotidesrequiredto
fuel glycosylation reactions have never been studied inͲdepth in wellͲsynchronized asexual
parasites or in other stages of its life cycle. These metabolites are of critical importance,
especiallyconsideringtherenewedinterestinthepresenceofNͲ,OͲandotherglycansinkey
parasite proteins. In this work, we adapted a liquid chromatography tandem mass
spectrometry method based on the use of porous graphitic carbon (PGC) columns and MSͲ
friendly solvents to quantify sugar nucleotides in the malaria parasite. We report on the
thorough quantification of the pools of these metabolites throughout the intraerythrocytic
cycle of P. falciparum. The sensitivity of the method enabled for the first time the targeted
analysisoftheseglycosylationprecursorsingametocytes,theparasitesexualstagesthatare
transmissibletothemosquitovector.
Summarystatement
APGCͲbasedLCͲMS/MSmethodhasbeendeployedforthequantificationofsugarnucleotides
inthemalariaparasite.Plasmodiumfalciparumgametocytesdisplayremarkablyhighlevelsof
somesugarnucleotides,comparabletotheextracellularformsofotherprotozoanparasites.
Shorttitle
ThesugarnucleotidepoolsofPlasmodiumfalciparumsexualstageparasites
Keywords
Glycobiology,Parasitology,Malaria,SugarNucleotides,Plasmodiumfalciparum,Gametocytes
2
Abbreviationsused
ESI, electrospray ionization; GDPͲFuc, GDPͲfucose; GDPͲGlc, GDPͲglucose; GDPͲMan, GDPͲ
mannose;GPI,glycosylphosphatidylinositol;GIPL,glycoinositolphospholipid;LCͲMS/MS,liquid
chromatography tandem mass spectrometry; MSPͲ1, merozoite surface protein 1; MSPͲ2,
merozoite surface protein 2; MRM, multiple reaction monitoring; PGC, porous graphitic
carbon;RP,reversedphase;SPE,solidphaseenrichment;TSR,thrombospondintype1repeat;
UDPͲGal, UDPͲgalactose; UDPͲGlc, UDPͲglucose; UDPͲGlcNAc, UDPͲNͲacetylglucosamine;
uRBC,uninfectedredbloodcell.
3
Introduction:
Plasmodiumfalciparumisthecausativeagentofthedeadliestformofhumanmalaria
anditisresponsibleforthemajorityofthemalariaͲrelateddeaths[1].Withapproximatelyhalf
oftheworldlivingatriskofmalaria,theemergenceofparasiteresistancetothecurrentlastͲ
lineofantimalarialsposesaseriousthreattomalariacontrolandeliminationefforts[2].The
developmentofnoveldrugsisstilllimitedbyourincompleteunderstandingofthebiologyof
theparasite[3].Thecharacterizationofparasitemetabolicpathways,newproteinfunctions,
postͲtranslational modifications and the identification of cognate receptors on human and
vectorhostcellscanultimatelyguidethedesignofselectiveinhibitorstopreventdiseaseand
parasitetransmissionthroughthevector.
ThefrequencyofglycosylationmodificationsforP.falciparumisgenerallyassumedto
be low in the blood stages of the parasite [4,5] except for the wellͲdescribed
glycosylphosphatidylinositol (GPI) anchors [6]. GPI anchors attach essential proteins, such as
merozoitesurfaceproteins1and2(MSPͲ1andMSPͲ2)[7],totheparasiteplasmamembrane.
Following parasite egress from the red cell, GPI anchors are also thought to induce
proinflamatory cytokine responses, which are a major contributing factor to malaria
pathogenesis[8].Recentadvancesintheidentificationofnewglycosylatedstructuresbothin
thehuman[9]andthemosquitostages[10],andthequantificationofsugarnucleotidesinthe
bloodstagesofparasitedevelopment[11]haverenewedtheinterestintheglycobiologyofP.
falciparum. Sugar nucleotides are high energy donors that feed the biosynthesis of
glycoconjugates [12]. For instance, GDPͲmannose (GDPͲMan) and UDPͲNͲacetylglucosamine
(UDPͲGlcNAc) are required for the synthesis of GPI anchors in eukaryotes [13,14]. Similarly,
UDPͲGlcNAcisalsoinvolvedinthesynthesisofNͲglycans,whichinP.falciparumconsistofone
ortworesiduesofNͲacetylͲglucosamine(GlcNAc)[9,15].Asobligateglycosylationprecursors,
quantification of sugar nucleotide pools provide strong evidence of the presence of cellular
glycosylation reactions [11,16]. Furthermore, the ability to identify and quantify these
4
metabolites remains an invaluable tool for functional analyses aimed at elucidating the
mechanisms of sugar nucleotide biosynthesis [17–19]. However, a robust analytical method
must take into consideration their highly polar nature, low in vivo concentration and rapid
turnover.Moreover,structurallyanalogoussugarnucleotides,suchasUDPͲglucose(UDPͲGlc)
andUDPͲgalactose(UDPͲGal),displaysimilarchromatographicprofiles,hinderinganadequate
resolution.
Sugar nucleotide pools of P. falciparum and other protozoan parasites have been
previously quantified by ionͲpair reversedͲphase (RP) liquid chromatographyͲtandem mass
spectrometry (LCͲMS/MS) [11,16]. In this approach, the weak retention displayed by polar
compounds under RP conditions is solved with the addition of ionͲpairing solvents. Still, this
strategyshouldbeavoidedwhentheinstrumentationisintendedtobeemployedindifferent
applications,sincetraceamountsofionͲpairreagentscannotbefullyremoved[20].Moreover,
thesereagentsreducesensitivitybypartiallysuppressingelectrosprayionization(ESI)[21,22].
In contrast to conventional columns used for RP chromatography, porous graphitic carbon
(PGC)notonlyretainsnonͲpolarcompoundsbutalsoexhibitsastrongaffinitytowardspolar
analytes.Thus,PGCisanexcellentchoiceformultipurposeMSinstrumentation(suchasthose
housed in a shared facility) since it does not require the addition of ionͲpair reagents to
achieveanefficientretentionofsugarnucleotides[20,23].Additionally,thecomplexretention
mechanism displayed by PGC results in an increased selectivity with respect to other
approaches;allowingtheseparationofstructurallysimilaranalytes[24].
In this work we used a PGCͲbased LCͲMS/MS technique [20] to identify and quantify
thesugarnucleotidepoolsintightlysynchronizedP.falciparumparasites.Differentstagesof
intraerythrocyticdevelopmentwereanalyzed,resultingingreaterprecisionintheestimatesof
availabilityorabundanceoftheseobligatedonorsforglycosylationreactions.Thesensitivityof
the analytical method allowed us to compare the sugar nucleotide content of gametocytes,
which are intraerythrocytic, nonͲproliferative single parasite forms, to the multinucleated
5
erythrocytic schizonts containing between 12Ͳ18 merozoites. The P. falciparum gametocyte
makes up less than 1% of the entire parasite biomass in an infected individual, retains a
unique,falciformshape,sequestersinthehematopoieticsystemofhumanbonemarrowand
doesnotegressfromitsredcellhomeuntilitisinsidethemosquitomidgut[25,26].Giventhis
known biology, previous works suggested that gametocytes entered a quiescent phase
associated to a lowͲmetabolic activity [27]. Thus, it was thought that sugar nucleotide
biosynthesis in gametocytes would be lower (or even absent) in comparison to the more
dynamic asexual blood stages. Nevertheless, our results seem to show that in fact the
converseistrue,inagreementwithrecentstudiesthatindicateasignificantmetabolicactivity
inP.falciparumgametocytes[28].
Experimental
Chemicals.Allreagents,standardsandsolventsusedforchromatography(HPLCgrade)were
purchasedfromSigmaͲAldrich(St.Louis,MO,USA),unlessotherwisespecified.Ammoniaand
absolute ethanol were purchased from Merck (Darmstadt, Germany). High purity water was
prepared with a Millipore MilliͲQ Plus system (Millipore, Bedford, MA, USA). Standard stock
solutionsofsugarnucleotideswerepreparedinwaterat2μMandstoredatͲ80oCpriortouse.
Further dilutions were prepared in 25% acetonitrile + formate buffer (0.1 % formic acid
adjustedtopH9withammonia).
P. falciparum culture and synchronization. P. falciparum asexual stages were cultured in an
atmosphereof92%N2,3%O2and5%CO2 withB+humanerythrocytesat2Ͳ4%haematocritin
RPMImediumsupplementedwith0.5%AlbumaxII.Humanerythrocyteswerepurchasedfrom
theBancdeSangiTeixits(Catalonia,Spain),afterapprovalfromtheComitèÈticInvestigació
Clínica Hospital Clínic de Barcelona. For the analysis of asexual blood stages, P. falciparum
strain 3D7 (BEI Resources/MR4/American Type Culture Collection, Manassas, VA, USA) was
6
tightly synchronized to a 5Ͳhour window by combining Percoll purification of schizonts [29]
followedbysorbitollysis5hourslater[30].Toreducebiologicalvariation,asingleculturewas
splitandparasiteswereharvestedat18,25,32,35and37±2.5hourspostͲinvasion.
Gametocytogenesis. The previously described 3D7ͲB subclone E5 [31] was cultured in RPMI
medium supplemented with 10% human serum for gametocyte production. Sorbitol
synchronizedrings(day0)weretreatedwith50mMNͲacetylglucosamine(GlcNAc)[32]that
was maintained for the remainder of the experiment. On day 10, asexual late stages were
removed by sorbitol lysis and gametocytes (mostly stage IV) were enriched from uninfected
redbloodcells(uRBCs)andresidualringstagesbymagneticcellsortingusingMACScolumns
(MiltenyiBiotech,Germany).
Metabolite extraction and preanalytical purification. Harvested parasites were released by
osmoticlysisin60volumesofcolderythrocytelysisbuffer(from10xstocksolutionconsisting
of 0.15M NH4Cl, 0.1M KHCO3, 0.01M EDTA) and incubated in ice for 10 minutes [33]. As a
control,4x109uRBCsweresubjectedtothesametreatment.Hostredbloodcellmaterialwas
washedthricewithiceͲcoldphosphateͲbufferedsalineandreleasedparasiteswerelysedwith
500 μLs of Ͳ20°C 70% ethanol and spiked with 20 pmols of internal standard GDPͲglucose
(GDPͲGlc). Cell debris wasremoved by centrifugation (20,000 x g for 10 minutes at 4ºC) and
supernatantsweredriedundernitrogenorinaSavantTMSPD1010SpeedVacTMconcentrator
(Thermo Scientific) at room temperature. For comparison purposes, some samples were
further processed to reduce complexity. Briefly, lipids were extracted by nͲbutanol:water
partition, and sugar nucleotides enriched by solid phase enrichment (SPE) with SupelcleanTM
EnviTMͲCarb columns (Supelco), as described previously [16,34]. Extracted metabolites were
finallyresuspendedin80Ͳ100μLsof25%acetonitrile+0.1%formicacidadjustedtopH9with
ammonia.10μLswereinjectedforLCͲMS/MSanalysis.
7
LiquidchromatographyͲelectrosprayionizationͲtandemmassspectrometry(LCͲMS/MS).The
LCͲMS/MS method was carried out on an Agilent 1290 Infinity LC System equipped with a
Hypercarb PGC column (5 μm, 2.1 x 100mm; ThermoFisher Scientific) coupled by ESI to a
QTRAP6500(ABSciex).Thecolumnwasmaintainedat60ºCandsamples(10μLvolume)were
injectedataconstantflowrateof150μL/min.Thebinarymobilephasewascomposedof0.1%
formicacidadjustedtopH9withammonia(A)andacetonitrile(B).TheLCrunstartedwitha
30 minute gradient from 2% acetonitrile to 15%, ramping linearly to 50% at minute 42 and
followedbya1minutegradientto100%,whichwasmaintainedfor2minutes.Beforerunning
the next sample, the column was reͲequilibrated by returning to the initial conditions of 2%
acetonitrilefor35minutes(Fig.S1).Themassspectrometerwasoperatedinthenegativeion
mode with the following settings: ion spray voltage: Ͳ4200V, declustering potential: Ͳ80V,
entrancepotential:Ͳ10V,sourcetemperature:600ºC.Parentandfragmentionsweredetected
in multiple reaction monitoring (MRM) mode for mass transitions described in Table 1. Peak
identitieswereverifiedbytheanalysisofcommercialsugarnucleotidestandardsandselected
areaswereintegratedwiththeAnalyst®1.6.2software.Sugarnucleotideswerequantifiedby
correlating the known amount of commercial sugar nucleotides in the calibration curve and
theratiobetweenmetaboliteandinternalstandardMSsignals.
Results
Validation of a PGC chromatography LCͲMS/MS method for the quantification of sugar
nucleotides in asexual P. falciparum blood stages. Commercial sugar nucleotide standards
were subjected to chromatographic separation on a PGC column and identified by their
characteristicelutiontimeswhenMRMfailedtoselectbetweenanalyzedcompoundsasinthe
caseofstereoisomers,suchasUDPͲGlcandUDPͲGalorGDPͲManandGDPͲGlc(Fig.1A).Allof
the standards could be well resolved to baseline. Linearity, limits of detection and
8
quantificationandintraͲdayandinterͲdayvariabilityofretentiontimesareincludedinTable1.
PreviouslydescribedmethodsinvolvedapreanalyticalpurificationstepconsistingofbutanͲ1Ͳ
ol[16]delipidationandSPE[16,20,34]toreducesamplecomplexity.Toassesswhethertheuse
ofthissamplepreparationstepcouldbecircumventedinintraerythrocyticstages,7.3x107P.
falciparum3D7trophozoites(30±2.5hpi)wereanalyzedwithorwithoutdelipidationandSPE.
Sugar nucleotide levels displayed no significant difference between both sample treatments
(Fig.1B),allowingforafasterandlesslaboriousmethod.Furthermore,backgroundsignalfor
each MRM transition remained unaltered and thus the low limits of quantification observed
using standards were maintained (Table S1). Remarkably, under these conditions we did not
observeanydecreaseinthechromatographicperformanceofthecolumn(i.e.peakresolution
oftheanalytes)inmorethan5differentrunswithseveralsamplesoveraperiodofmorethan
ayear.
Quantifying sugar nucleotide levels at different stages of P. falciparum asexual
intraerythrocytic development. In its human host,P. falciparumgoes through a cycle of
repeatedinvasionoferythrocytes,exhibitingasynchronyofabout48Ͳhduration.Inculture,P.
falciparumgrowninculturelosessynchronicityquickly,makingitmoredifficulttoanalyzeand
compare sugar nucleotide levels across specific intraerythrocytic developmental stages.
Different synchronization methods, based on selective lysis [30] or physical separation of
specific stages [29] generate synchronization windows as wide as the duration of that stage,
whichisnotoptimaltothoroughlydescribetheregulationofspecificmetabolitesthroughout
the parasite intraerythrocytic life cycle. Higher degrees of synchrony can be achieved by
successivecombinationofdifferentmethods,suchasPercollpurificationfollowedbysorbitol
lysis [35]. However, these procedures require significant amounts of culture reagents or the
reduction of the quantity of parasites in culture, making more difficult the analysis of lowͲ
abundantmetabolites.Thelowquantificationlimits(0.8Ͳ2.6nM;Table1)achievedbythisLCͲ
9
MS/MSmethodpromptedustoanalyzetightlysynchronizedparasitecultures(0Ͳ5hwindow)
with~1Ͳ4x108parasitescells.Theseanalysesresultedinacomprehensivequantificationofthe
sugar nucleotide levels along the in vitro asexual intraerythrocytic development of P.
falciparum(Fig.2B).
Oneof the mainissues whenanalyzingtheintraerythrocyticstagesofthe malariaand other
intracellular parasites is the extent of hostͲcell contamination. Human red blood cells (RBCs)
contain pools of sugar nucleotides (Fig. S2A) and the LCͲMS/MS method could detect
significant amounts of these metabolites carried over from the parasite host cells. To assess
thedegreeofthiscontamination,weanalyzedthesugarnucleotidesdetectedinlyseduRBCs
and established a contamination threshold that may be carried over from the host cell (see
dashedlinesinFig.2B).OnlyGDPͲFuc,UDPͲGlcandUDPͲGlcNAcweredetectedabovethelimit
of quantification when high numbers of uRBCs (a4x109) were lysed (Fig. S2B). Nevertheless,
even in that case the background levels were negligible when compared with sugar
nucleotidesdetectedinparasitesinthetrophozoitestagesandlater(from32±2.5hpi,seeFig.
2B).
Sugar nucleotide levels in sexual gametocytes of P. falciparum. After establishing infection,
less than 1% of intraerythrocytic P. falciparum differentiate to gametocytes [36].
Gametocytogenesisisanessentialprocessforparasitetransmission,sincegametocytesarethe
only stages that can fertilize and establish the sporogonic cycle in the anopheline mosquito
vector. Recent metabolomic studies reveal that despite entering a nonͲproliferative state,
gametocytesexhibitincreasedlevelsofglucoseutilizationandhighenergyrequirements[28],
indicating significant metabolic activity in these life stages. The gametocytes are known to
harbor many transcripts while in the human host that are rapidly translated into proteins
following gametogenesis in the mosquito, which occurs within a 10Ͳ15 minute time period,
followed by macrogamete fertilization to form the mosquitoͲinfective ookinete stage.
10
FertilizedzygotesandmotileookinetesexpresstwomajorGPIͲanchoredproteins,P25andP28
[37].WehypothesizethatsugarnucleotidepoolsarerampedͲupandstoredinpreparationfor
gametogenesis inside the mosquito vector. To date, the pools of sugar nucleotides in
gametocytes have not been analyzed. The sensitivity of the HPLCͲMS/MS method designed
enabledtherobustanalysisofsamplescontaininglessthan1x108P.falciparumgametocytes
(seeTableS2).Weobservedthatsomeofthesugarnucleotidesdetectedinthislifestagewere
presentatmuchhigherlevelsthanintheasexualbloodstages(Fig.3B).
Discussion
In this work we used a LCͲESIͲMS/MSͲbased methodology for the quantification of sugar
nucleotideintheintraerythrocyticstagesofP.falciparumusinganiontrapinstrument(QTRAP
6500 AB Sciex), a PGC column and a MS compatible solvent system [20]. The low limit of
detectionforthetargetedanalytes,between0.3Ͳ0.8nM,allowsforareductioninthenumber
ofparasitesrequiredforanalysis(seeTableS2).Thisiscriticalwhenworkingwithorganisms
such as P. falciparum, which poses significant problems for obtaining sufficient amounts of
parasites that are tightly synchronized or from specific life stages (i.e. gametocytes, or
ookinetes). The analysis showed (1) a dramatic increase of the pools of different sugar
nucleotidesinthematurestagesoftheintraerythrocyticasexualdevelopmentoftheparasite,
and(2)remarkablyhigherlevelsofthesemetabolitesinstageIVgametocytes.Weanticipate
leveraging the sensitivity of this method to analyze sugar nucleotides in other important
parasitelifestages,suchastheintrahepaticstagesofP.falciparum,whichislessamenablefor
largeͲscalepurification.
Aspreviouslyobserved[11],theavailabilityofsugarnucleotidesintheringstagesseemstobe
scarce(Fig.2B),suggestingthatglycosylationreactionsmightoccurlaterintheparasitecycle.
However,itcouldalsobearguedthatthehighdemandofthesemetabolitesduringtheinitial
stageskeepsthepoolsatalowerlevel.Throughoutasexualdevelopment,thereisasignificant
increaseinparasitesizewithintheredbloodcell[38].Itsrateofmultiplication,with10to20
11
merozoitesproducedevery48h,involveshighmetabolicandbiosyntheticactivity.Theparasite
assemblesavarietyofglycoconjugates,mostlyfreeGPIsorglycoinositolphospholipids(GIPLs),
andGPIͲanchoredproteins,severalofwhichareessentialforinvasion[6,39,40].Regardlessof
thepresenceofNͲglycans[9]andotheruncharacterizedglycosylations[19],GPIbiosynthesis
requires UDPͲGlcNAc and GDPͲMan donors [41]. Accordingly, the pools of these metabolites
(Fig.2B),butalsoothersugarnucleotides,expandexponentiallyalongtheasexuallifecycleof
theparasite,eitherreflectinganincreasedbiosynthesistofuelthedemandofUDPͲGlcNAcand
GDPͲMan during the mature stages of the asexual life cycle; or indicating a decrease on the
need of these metabolites, due to a decline of glycoconjugate synthesis at the final stages.
Data on gene expression of UDPͲNͲacetylglucosamine pyrophosphorylase and mannoseͲ1Ͳ
phosphateguanyltransferasemightshedmorelightintothismatter,buttheirRNAtranscripts
aredecreasedandincreased,respectively,towardstheendofthelifecycle[42].
ThelevelsofGDPͲfucose(GDPͲFuc)aresteadybetween32±2.5and35±2.5hourspostͲinvasion
suggesting a possible extra consumption of this metabolite by the parasite glycosylation
machineryduringthistimeperiod(Fig.2B).Interestingly,P.falciparumisknowntoexpressa
ProteinOͲfucosyltransferase2(PoFUT2)homolog,aGDPͲFucͲdependentOͲfucosyltransferase
that fucosylates thrombospondin type 1 repeat (TSR) domains in other organisms [43].
Accordingly, the transcription of PfPoFUT2 and potential OͲfucose acceptors, the TSRͲ
containingproteinsMTRAPandTRAMP,increaseduringthatsameperiod[42].
Finally, gametocytes present significantly higher levels of sugar nucleotides than the asexual
bloodstages(Fig.3B).Theliteraturesuggeststhatthecellvolumeofgametocytesandasexual
schizontsissimilar[38].Therefore,theobservedlevelsofUDPͲGlcNAc,UDPͲGlcandGDPͲMan
above one order of magnitude in comparison to asexual parasites shows a much higher
concentration and availability of these metabolites in gametocytes (i.e. 65.7, 98.8 and 7.8
pmols/107 parasites cell for UDPͲGlc, UDPͲGlcNAc and GDPͲMan). Indeed, the levels of UDPͲ
GlcNAc, UDPͲGlc and GDPͲMan in gametocytes are comparable to the extracellular forms of
12
otherparasites,suchasTrypanosomabrucei,TrypanosomacruziorLeishmaniamajor[16]that
present more varied and profuse glycosylation patterns [44]. This could reflect a distinct
glycosylation status in P. falciparum gametocytes in contrast to asexual blood stages and
supports the contention that the gametocytes are preparing for an extracellular existence in
themosquito.Gametocytesegressfromtheredbloodcellandbecomeextracellulargametes
in the mosquito midgut, where they could make use of the pools accumulated in the
gametocytestage.Thus,glycosylationcouldbemorerelevantintheseextracellularformsand
possibly also in other mosquito stages (such as oocysts and sporozoites). Further studies on
theglycosylationspresentinthesurfaceofP.falciparumextracellularstageswillhelptocast
lightonthisissue.
Acknowledgements
The authors thank Hernando A. del Portillo and Alfred Cortés for support and helpful
discussionsthroughoutthisproject.WethankAlfredCortésandcolleaguesforthegenerous
giftofP.falciparumstrain3D7ͲBsubcloneE5usedingametocytestudies.Wearealsograteful
toM.Ramírez,M.Cova,S.SanzandCCiTUB(ScientificandTechnologicalCentersUniversitat
deBarcelona)fortechnicalsupport,advice,assistanceandusefulsuggestions.
Declarationsofinterest
Theauthorsdeclarethatnoconflictofinterestexists.
Fundinginformation
LIthanksfundingfromSpanishMinistryofEconomy(grantSAF2013Ͳ43656ͲRsupportingBLͲG).
LI and RRD are grateful for National Institutes of Health grant 1R21AI115063Ͳ01. LI is a
member of the GlycoParͲEU FP7 funded Marie Curie Initial Training Network (GA 608295).
ISGlobalissupportedbytheCERCAProgramme(GeneralitatdeCatalunya).
13
Authorcontributionstatement
BLͲG collected samples and carried out sugar nucleotide analysis. BLͲG and LI conceived the
work, designed the method and performed data analysis. BLͲG, LI and RRD outlined the
documentandcontributedtothewritingandreviewofthismanuscript.
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17
565ї323
606ї385
604ї424
604ї362
588ї442
UDPͲGlc
UDPͲGlcNAc
GDPͲMan
GDPͲGlc
GDPͲFuc
34.21
33.57
31.18
22.40
21.68
20.38
tR
(min)
1.04
1.22
1.94
1.54
1.51
1.57
tRintraͲdaySD
(min)b
2.90
2.57
2.95
3.11
3.44
2.21
tRinterͲdaySD
(min)c
0.44
0.20
0.77
0.30
0.77
0.25
LoD
(nM)d
0.84
0.68
2.56
2.56
1.48
1.02
LoQ
(nM)e
0.9985
1.0000
0.9966
0.9973
0.9987
0.9968
f
R2
18
a.Themajorproductionforeverysugarnucleotidecorrespondstoitsnucleotidicmoiety,sinceionfragmentationtakesplacearoundtheunstablebondbetweenthesugar
andthenucleotide(i.e.atthesugarͲ1Ͳphosphateorpyrophosphatelinkages)[16].
b.IntraͲdaystandarddeviationofretentiontimes(n=12measurements).
c.InterͲdaystandarddeviationofretentiontimes(n=3measurements).
d.Limitofdetection(LoD)wascalculatedatasignalͲtoͲnoiseratio(S/N)of3.
e.Limitofquantification(LoQ)wascalculatedatasignalͲtoͲnoiseratio(S/N)of10.
f.CorrelationcoefficientsoftheknownamountofcommercialsugarnucleotidesinthecalibrationcurveandtheanalyteMSsignalrelativetotheGDPͲglucoseinternal
standard.
565ї323
MRMtransition
(m/z)a
UDPͲGal
Analyte
Table1.Retentiontimes,limitsofdetectionandquantificationandlinearityofstandardcalibrationcurves
Figurelegends
Figure 1. Development of the PGC LCͲMS/MS quantification method for sugar nucleotides
analysis.(A)Representativeextractedionchromatograms(XIC)oftargetedsugarnucleotides
fromanextractofP.falciparum3D7trophozoites(30±2.5hpi).(B)Sugarnucleotidepoolsof
7.3x107P.falciparum3D7trophozoites(30±2.5hpi)analyzedbyPGCLCͲMS/MSbefore(white
bars)orafter(greybars)apreanalyticalpurificationconsistingofbutanͲ1Ͳoldelipidationand
solidphaseenrichment.Valuesofeachsugarnucleotideindicatetheaverageofthreedifferent
extractions.ErrorbarsdisplayS.D.
Figure 2. Sugar nucleotide pools of P. falciparum asexual blood stages. (A) Schematic
representationofthesynchronizationstrategyandsamplingtimeͲpoints.(B)Quantificationof
sugarnucleotidesfromextractsofP.falciparum3D7tightlysynchronizedasexualbloodstages.
Values are the average of three extractions at different timeͲpoints. Error bars display S.D.
Uninfected red blood cells were lysed as a negative control and were used to estimate the
maximumcontaminationthresholdexpectedfromhostredbloodcells(dashedline)according
totheamountofredbloodcellsineachsample(exceptfornonͲquantifiableanalyteswitha
signalͲtoͲnoiseratio<10,i.e.UDPͲGalandGDPͲMan).
Figure3.SugarnucleotidepoolsofP.falciparumgametocytes.(A)Schematicrepresentation
ofgametocyteinductionandsamplingtimeͲpoint.(B)Quantificationofsugarnucleotidesfrom
an extract of P. falciparum 3D7B E5 subclone. Values are the average of three technical
replicates.ErrorbarsdisplayS.D.
19
Fig. 1
A.
UDP-Glc
100%
565 ї 323
Relative intensity
UDP-Gal
0%
100%
606 ї 385
UDP-GlcNAc
0%
100%
GDP-Glc
604 ї 424
GDP-Fuc
588 ї 442
GDP-Man
0%
100%
0%
0
pmols/1x107 parasite cells
B.
20
0,6
0,4
0,2
0,0
40
Time (min)
60
80
Fig. 2
A.
B.
37±2.5 hpi
0,4
32±2.5 hpi
35±2.5 hpi
25±2.5 hpi
18±2.5 hpi
pmols/107 parasite cells
0,3
UDP-Gal
3,0
UDP-Glc
3,0
2,0
2,0
1,0
1,0
UDP-GlcNAc
0,2
0,1
0,0
0,8
0,6
0,0
0,0
15 20 25 30 35 40
GDP-Man
15 20 25 30 35 40
0,3
GDP-Fuc
0,2
0,4
0,1
0,2
0,0
0,0
15 20 25 30 35 40
15 20 25 30 35 40
Time post-invasion (hours)
15 20 25 30 35 40
Fig. 3
A.
STAGE I
(Day 3)
LC-MS/MS
(Day 10)
STAGE III
(Day
ay 6)
GlcNAc
induction
MACS +
sorbitol
RING STAGE
(Day 0)
pmols/1x107 cells
B.
STAGE II
(Day 4)
STAGE IV
(Day 8)
125
0.40
100
0.20
75
0.00
50
25
0