462 Chimia 2013, 67, Nr. 7/8 analytical sciences

doi:10.2533/chimia.2013.462 Chimia 67 (2013) 462–475 © Schweizerische Chemische Gesellschaft

Analytical Sciences As01 Analytical Sciences As02

ESTASI Evaluation of Standardsfor Quantitative TissueImaging by LA-ICP-MS Elena Tobolkina, Natalia Gasilova, Liang Qiao, Qiuliyang Yu,Hubert H. Girault DanielA.Frick,1 Charlotte Giesen,2 TeresaHemmerle,3 DarioNeri,3 Bernd Bodenmiller,2 DetlefGünther1 Laboratoire d’Electrochimie PhysiqueetAnalytique, Station 6, EPFL, CH- 1015 Lausanne 1Department of Chemistryand AppliedBiosciences,Laboratoryof InorganicChemistry, ETHZürich, CH-8093 Zürich 2Institute of MolecularLifeSciences, University of Zürich, CH-8057 Zürich 3Department of Chemistryand AppliedBiosciences,Institute of In thislecture,weshall recall some electrochemicalconcepts underlying Pharmaceutical Sciences,ETH Zürich, CH-8093 Zürich ionization methods for such as MALDI and ESI. Then,weshall presentanovel contactless electrostatic spray methodrecent- Over thelastfew years, imaging of softtissuestodeterminethe spatially ly developed that can be used to spray from sample droplets depositedonan resolvedconcentrations or isotope ratioshas emerged sincethe firstsuccess- inertpolymer substrate or fromsamples in aglass capillary. Thegist of the fullaser ablationofsofttissues. [1]Recently asecond direction, apart from method is to chargeelectrostatically the surface of the liquid untilthe elec- thedeterminationofthe elementconcentrationhas calledattention: quanti- trostaticpressure overcomes the interfacial tension to emit charged droplets. tativeimmunoassay using elementlabeled antibodies.[2] With this tech- nique multiple antigenscan be marked on thesametissue sample.Bycom- With this method,weshallshow how to spray proteins or peptides being bining thehighspatialresolutionofthe laserablationset-upconnected to separated in apHgradient gel by isoelectric focusing. Thisapproachpro- thehighsensitivity of an ICP-MS this allows efficient multiplexing to sam- vides adirect mass spectrometryreadout of IEF gels. plethe currentstatusofcells within atissue section.

Then,weshallshow how it is possible to spray from spy-holes in microflu- QuantitativeLA-ICP-MS reliesonsuitableinternaland external standards; idic chips where aqueoussolutionsare being processed. This concept is fur- internal standardstocorrectfor driftofthe instrument,sensitivity differ- therappliedtodroplet-fluidicreactors. encesand differencesofthe samplesand external standardsfor assessing the concentration. Thisworkinvestigates suitable internal standardsfor tissue imaging to correct forablated mass, transport andionisationinthe ICP. Ad- ditionally acomparativeapproach forthe quantificationusing external standardswill be shown. [1] LiangQiao, Romain Sartor,Natalia Gasilova, Yu Lu, Elena Tobolkina, Baohong Liu, and Hubert HGirault, Anal. Chem,. 2012, 84,7422. [1]P.Ek, presentedinpartat1998 Winter Conference on Plasma Spectro- [2] Liang Qiao, Elena Tobolkina, Baohong Liu, and Hubert HGirault, , Scottsdale,AZ, USA, 05 -10January, 1998. Anal. Chem, 2013,inpress, DOI: 10.1021/ac400472q [2]C.Giesen, L. Waentig,U.Panne,N.Jakubowski, Spectrochim.Acta, Part B, 2012, 76,27-39.

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Mass Spectrometry Based DisulfideAssignmentinVenom Proteins Developmentofanonlinesolid phase extraction system usingpolyether- etherketone tubing coupledtoion traptandemmass spectrometry Franka Kálmán, Marc E. Pfeifer, DenisPrim,Miriam S. Goyder XueLi1,2,RenatoZenobi2 Institute of Life Technologies, University of Applied Sciences Western Switzerland(HES-SO Valais), RouteduRawyl 64, 1950 Sion, Switzerland 1Shanghai University, Shanghai 200444, P. R. China 2ETH Zürich,CH-8093 Zürich,Switzerland

Disulfides play acrucial role in protein structure and function. Their charac- Effectiveabsorptionofhydroxylatedpolyaromatic hydrocarbons to poly- terization in therapeutic protein production is necessary to ensure active etheretherketone (PEEK)tubing hasbeenrecentlyreported, andbased on this, pharmaceutical ingredient (API) safety and efficacy. Massspectrometry an onlinesolid phase extraction(SPE)systemusing PEEK tubing forpre- (MS) can be used to characterize disulfidebridges in proteinstodetermine treatingurinary 1-hydroxypyrene(1-OHPyr)was developed(Fig. 1). PEEK tubing Sampling cone both thenumber and the connectivity of disulfides.[1]Venomscontain HPLC High voltage HO many disulfide-rich proteins that are potential drug candidates.[2] However, Pump 189 N2 MS based disulfideassignment is challenging forvenom proteins due to HPLC to MS 1-Hydroxypyrene Solvent their knotted disulfide structure. Here we demonstrate determination of di- 217 CO sulfide number and connectivity in Ribonuclease Aand Insulin using tech- Waste m/z niques including chemical cleavage, partial reduction, pepsin digestion and Fig. 1Schematic diagramofanonlineSPE system using PEEK tubing cou- collision induceddissociation. Furthermore, we show the application of pled to iontrap tandemmassspectrometry(MS/MS). techniques in MS to disulfide assignmentinthe three-fingertoxin, Mambin. EffectsofHPLCflowrateand PEEK tubing i.d. were studied(Fig. 2) to- gether with compound concentration, injectionvolumeand system stability, [1]Jeffrey J. Gorman, Tristan P. Wallis, James J. Pitt, Mass Spec. Revs. demonstratinggood analytical performance of theonlinePEEK-SPE system. 2002, 21,183. (a) (b)

5 ) ) 6.0x10 u. 0.5mL/min

[2] R. Manjunatha Kini, Toxicon 2010, 56,855. u. 40000 a. a.

i.d.0.25mm i.d.0.10mm y( y( i.d.0.13mm it it 4.0x105 ns ns

te 0.2mL/min 0.3mL/min te

in 20000 in

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te 2.0x10 lu lu so so Ab Ab 0.0 0 2x102 3x102 4x102 5x102 12345 Inner surfaceareaofPEEK tubings (mm2) Time (min) Fig. 2Effects of PEEK tubing i.d. andHPLCsolvent flow rateonabsorption anddesorptionof1-OHPyr to PEEK tubing. [1]X.Li, R. Zenobi, Anal.Chem. 2013, 85,3526. analytical sciences Chimia 2013, 67, Nr. 7/8 463

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Microwave Plasma –AtomicEmission : anovel Structural analysisofmonoclonal antibodies by top-down andmiddle- technology forthe analysisofimpuritiesingold down mass spectrometry

Thomas B. Jensen1,2,Jean-Pascal Bourgeois1,JonathanJ.Jodry2 Luca Fornelli, Ünige A. Laskay,Anton N. Kozhinov, Kristina Srzentić, Daniel Ayoub, YuryO.Tsybin 1Ecoled’ingénieursetd’architectes de Fribourg, Bd.dePérolles80, 1705 Fribourg, 2MetalorTechnologiesSA, Av.duVignoble, 2009 Neuchâtel École Polytechnique Fédérale, 1015, Lausanne,Switzerland

With thesteep increaseinthe prizeofgoldinrecent years adecreaseinthe Comprehensive structureanalysis of antibodies, andspecifically immuno- qualityofraw materialshas been observed in thegoldrefining industry. globulins G(IgGs), is indispensable due to their leadingrole as biotherapeu- Specifically,highcontents of toxicelements(e.g. As,Cdand Se)are a tics. We recentlypresented encouraging resultsobtainedbyhigh-resolution serious health andenvironmentconcern.Thisincreasesthe need for OrbitrapFourier transform(FT)mass spectrometry(MS) in characterizing constantdevelopmentofanalytical methods. IgGs in atop-down fashion, i.e. with gas-phasefragmentation of the intact ~150 kDa proteinsbyelectron transferdissociation (ETD).Withthistech- Microwave Plasma –Atomic Emission Spectroscopy(MP-AES) is anovel nique ~32% sequencecoveragewas reached,but with incomplete sequenc- technologyfor elementalanalysis. The nitrogen plasma is an ing of complementaritydetermining regions (CDRs), responsible for the environmentallyfriendlyalternativetocurrentlyusedtechniques IgGaffinity andspecificity. We thus applied amiddle-down approach, (InductivelyCoupled Plasma –Optical EmissionsSpectroscopyand Atomic fragmentingIgGs in solution by proteolyticcleavage with IdeS (immuno- Absorption Spectroscopy), eliminatingthe need forexpensive and globulin G-degradingenzymeofStreptococcuspyogenes), andreducing flammablegasses. Furthermore, nitrogen can be generated locallyfrom disulfide bonds. The resulting three~25 kDacysteine-reduced fragments atmosphericair,avoiding logistical problemsofsupplying gascylinders to (Fd, Fc/2, andlight chain) were separated by reverse-phase mines in remotelocations. (C4 column) andsubjected to ETD tandemMS(MS/MS).Contrary to con- ventionalapproaches,original transient signalsfromseparatechromato- We have usedMP-AEStoanalyse thecontents of 15 elements in samples graphic runs were acquired, summed,and processed using FT or in-house fromall overthe world. The results were compared with values previously implemented filter diagonalizationmethod (FDM). Thus, the overallquality determined with ICP-OESand found to be in excellent agreement. of the experimental data wassubstantiallyimproved. MS/MSspectra were Moreover, thelinear range of thesignaloverseveral orders of magnitude analyzed with commercial software andvalidated manually. Thecombina- reduces theneed forrepeatedanalysesofsolutions with different dilutions. tion of highlyspecific digestion andreduction of disulfide bonds allowed the MS characterizationofvariable domains of both the lightand heavy chains, includingthe CDR2 region. Developed LC-MS/MS methods areof direct interestfor quality control of monoclonalantibodies in pharmaceuti- cal industry, as well as for furthermethod development toward MS-based structure analysis of mixturesofantibodies andantibody-drug conjugates in drug discovery.

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Advances in azide-alkynecycloaddition (click-chemistry) based manu- Fluorescence Resonance Energy Transfer of Gas-Phase Ions under facturingofmolecular diagnostic peptidemicroarrays Ambient Conditions

Denis Prim,VincentCosandey, Fabien Rebeaudand Marc E. Pfeifer Fanny Widjaja, Vladimir Frankevich, Konstantin Barylyuk, Zhiyi Yang,and Renato Zenobi InstituteofLife Technologies,HES-SOValais Route du Rawyl64, 1950Sion ETH Zurich, Department of Chemistry and Applied Biosciences, Wolfgang Pauli Str. 10, 8093 Zurich, Switzerland Rapid andreliable functionalizationsofsolidsurfaces areprerequisites for the efficient manufacturingofpeptide microarraysfor in-vitro diagnostic The use of mass spectrometry to study biomolecules in the gas phase has (IVD)applications.Whenworking with biological samples suchasserum been greatly enhanced due to development of soft ionization methods such non-specific binding (NSB) of complex matrix components to the spotted as MALDI and ESI. However, the link between the structures and confor- peptide probesand/or surrounding area must be limited in ordertoattaingood analytical performances. mation of biomolecules in solution and in the gas phase remains unknown. Previously, Fluorescence Resonance Energy Transfer(FRET)has been used Here we present resultsonbinding kinetics of various azide derivatizedpep- to probe conformationsofgaseous polyproline-based peptides inside a tidestoaza-dibenzocyclooctyne (ADIBO) activated surfaces basedoncop- quadrupole trap mass spectrometer. This opened apromising route to per-freeclick chemistry. Furthermorespecificantibodytargetmolecule cap- probe the conformations of large gaseous ions. Nevertheless, it requires ex- turing efficiencies were investigated andanalytical sensitivities compared to tensive instrument modifications, and the fluorescence intensity depends on standard microtiter plate basedinverse ELISAformats. the number of ions trapped inside the mass spectrometer. In the present study, we employed alaser-induced fluorescence technique directly in an Aselection of azide-tagged“blocking reagents”ofprotein,polysaccharide, ESI plume to monitor the FRET dynamics of isolated ions as the solvent polyethylenglycol (PEG) andaromaticcompound origin were testedfor their are removed from the system. This study opens up aroute for efficacy in preventing detrimentalNSB whenworking with serum.Infact, direct correlation between the structures and conformation of biomolecular one of the blocking reagents revealed an exceptionally strong capacity to in- ion in the gas phase and in solution without the need of ion trapping and hibitserum components to interactwith themicroarraysurface. complex instrumentation.

To conclude, compatibility of reagentaspectssuchasreactivities, sensitivi- ties,reproducibilities andblocking efficiencies withanautomated nano-liter scalemicroarrayspotting instrument were demonstrated with c-mycand BRCA-associated ring domain protein 1(BARD1) cancer diagnostic model assays. 464 Chimia 2013, 67, Nr. 7/8 analytical sciences

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Utility of chemical agents forhigh-throughput proteincharacterization AComparisonofNMR Predictors UsingBinaryTreeSimilarityofSpectra Kristina Srzentić 1;ÜnigeA.Laskay 1;YuryO.Tsybin1 AndrésM.Castillo, Julien Wist, LucPatiny, AndrésBernal 1Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Facultad de Ingeniería,Universidad Nacional de Colombia, BogotáD.C., Mass spectrometry(MS)-based proteomics,orhigh-throughput characteri- Colombia zation of proteins constitutes acomprehensive analysis of proteomesofdi- verse organisms. ConventionalMS-basedproteomics relies on protein en- Chemistry Department, Universidaddel Valle, A.A. 25360 Cali,Valle, zymaticdigestion intoshort, 0.6-3 kDa, peptidesprior to MS analysis.Re- Colombia. centadvances in MS instrumentation provide the necessary platformfor InstituteofChemical Sciences andEngineering, Ecole Polytechnique investigating increasinglylonger(3-7kDa)peptides in ahigh-throughput Fédérale de Lausanne (EPFL),CH-1015 Lausanne,Switzerland. manner. Herein, we describe adirected samplepreparation workflow using Grupo de Química Teórica,Universidad NacionaldeColombia, Bogotá chemical cleavage, as opposed to the enzymaticdigestion. Chemical cleav- D.C., Colombia. ageallowstargeting of rare amino-acids, for whichnoproteolytic enzymes exist.Asaresult,the averagesizedistribution of the generated peptides The rooted tree method for NMRspectra comparisondeveloped recentlyby could be increased. With thisapproach we aimtoimprove high-throughput theauthors[1] wasappliedtodesign amethodology for comparison and proteome characterization. The averagesizedistribution of peptides, effi- evaluation of NMRpredictionalgorithms.For each predictionalgorithm, a ciencyofpeptide fragmentation methods andthe probabilityofidentifying matrixisconstructedbyevaluatingthe similarity betweenthe experimental post-translationalmodifications were investigatedusing acommercial 48 spectra of 1000 molecules(rows) andtheir corresponding predicted spectra protein mixture (UPS1,Sigma Aldrich).Consecutive Methionine (M)and (columns). Aquery of each experimentalspectrum to thedatabase of simu- Tryptophan (W)cleavageswereperformed with CNBr.BNPS-Skatolewas lated spectra is performed andNMR predictorsare rankedaccording to their used for selectivecleavage at Wboth individually,and afterpartial CNBr Mean Reciprocal Rank (MRR). This methodology dispensesuswith the cleavage of M. Hydroxylaminewas used to achieve selectivecleavageafter need of assigning each signalwith itsnucleus, which makesthe task of Asparagine (N). Cleavage at Nterminus of Cysteine (C) residueswas per- benchmarking NMRpredictors less tedious andavoids anyhuman mistakes. formed with NTCB.Resulting peptidesweredesaltedonC4and C18 We used thisapproach to comparefour popular NMR predictors(ACDlabs, ZipTipcartridges(Millipore,Billerica, MA),and separated on aC8nano- Modgraph, Chemdraw, Spinus) andfound that ACDlabsprovidesthe best LC column. Elutedpeptidesweresuperchargedpost-columnwith 0.5% m- resultsbyfar (0.6 MRRfor ACD, ~0.4 MRRfor theother predictors). It is NBAand analyzed usingOrbitrapEliteETD FTMS.Due to the longaver- worthnotingthatthe freeSpinus platform’s performance is comparable to agepeptide length, high mass resolution wasused for both MS andMS/MS that of theother commercial alternatives. Theseresults areconsistent with scanning (60,000 and15,000 at m/z 400, respectively). Sequencecoverage andprotein identificationscores were obtainedusing Sequest, Mascot, MS- thoseobtainedusing thetraditionalmethodology of comparing deviations Align+,OMSSA andX!Tandemdatabasesearch algorithmsand compared on predicted shifts relative to theexperimental ones. with resultsproduced by classical MS-based proteomics approaches. [1]CastilloA., UribeL., Patiny, L.,Wist, J., Chemometr.Intell. Lab. (sub- mitted)

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Fossil fuel analysis via high resolution mass spectrometry. Analysisofacidiccompounds by CE in negative ESI-MS with organic solvents Konstantin O. Zhurov,Anton N. Kozhinov, Yury O. Tsybin Grégoire Bonvin,Serge Rudaz, JulieSchappler Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland School of Pharmaceutical Sciences,UniversityofGeneva, Universityof Fossil fuelsare complexmixtures of moleculeswith mostly C H N O S c h n o S Lausanne,Bdd’Yvoy20, 1211 Geneva 4, Switzerland elementalcompositions. It is imperative to identify and quantify these mole- cules,asthey affect the physicaland chemical properties of the sample. For Non-Aqueous CapillaryElectrophoresis (NACE) is an attractive CE mode instance, napthenic acids can corrode refinery units; sulphur-containing that consists in replacing the waterofthe background electrolyte(BGE) by organic solvents. This substitution alters severalparameters such as pKa, compounds must be removedfrom fuels used in transport,toreduce envi- dielectric constant, viscosity, zeta potential andconductivityresulting in ronmental damage. Finally,metal porphyrins found in medium and heavy modificationofCEseparationperformance. In addition, the use of NACE is particularlywelladapted to ESI-MS due to the highvolatilityofsolvent and crudeoilsdegrade catalystsused in cracking and can form corrosivespecies. low generated currents. Organic solvents also permit to reducethe number As there aremany molecules with very small differences in masses, often- of side electrochemical reactions at the ESItip allowing astabilizationof ESIcurrent andadecrease of background noise. All these featuresmakethe timesless than 1.1 mDa,high resolutionand high mass accuracy mass spec- NACE an interesting alternative to theclassical CZEmode especiallyto trometers are required. We have thus developed an analytical platform, improve selectivity, sensitivity,and spraystability. The aimofthisworkwas to evaluatethe useofNACEinnegativeESI-MS whichfor the firsttime successfullyemploys the OrbitrapElite Fourier for the analysis of acidiccompounds with two interfaces (sheathliquid and transform mass spectrometer, to analyze complex fractions of fossil fuels. sheathless). In afirst attempt,the NACE mode wascompared to the classi- cal CZEmodefor theanalysis of pharmaceutical acidiccompounds The platform provides advantages in comparison with other approaches and (NSAIDs).Thenthe sensitivity andseparationperformanceachieved by introduces several new key features, including arecalibration method which both interfaceswereevaluatedaswellasthe impact of BGEand sample composition. Finally, analyses on glucuronidesinurine samples were per- provides the required mass accuracy, andadata visualization method for formed in ordertoevaluatequalitativelyand quantitativelythe matrix ef- sample fingerprinting and comparison. It has been tested on resin and mal- fects. tene fractions of crude oil. Results demonstrate that the platformcurrently allows for in-depth analysis of polarcomponents of crude oil fractions of lowtomediumcomplexity. analytical sciences Chimia 2013, 67, Nr. 7/8 465

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14Canalysis of carbonates at high spatial resolution by the coupling of Fluorescence SensingofSpermine LaserAblation with AMS KaySeverin, Ziya Köstereli C. Münsterer1,2,L.Wacker2,B.Hattendorf1,M.Christl2,J.Koch1,R. Dietiker1,H.-A. Synal2,D.Günther1 1Laboratory of InorganicChemistry, D-CHAB, ETHZ, Wolfgang-Pauli-Str. Institut desSciences et Ingénierie Chimiques, ÉcolePolytechniqueFédérale 10, 8093 Zurich, Switzerland de Lausanne(EPFL),Lausanne, Switzerland. Fax: +41(0)21 6939305; Tel: 2 Laboratory of Ion Beam Physics, ETHZ, Schafmattstr. 20, HPK, 8093 Zur- +41(0)21 6939302 E-mail: [email protected] ich, Switzerland Spermine is anaturalpolyamine that is found in eukaryoticcells andbody 14Cisanimportant isotope for dating carbonates such as speleothems,corals fluids. Monitoring spermine concentrations in urinewas proposedasatool and shells[1]. Measurements of chemically processed and graphitized sam- for earlycancerdiagnosis.1 Opticalmethods basedonfluorescenceare in- ples are laborious and the achievablespatial resolution is limited. By using teresting alternatives because measurements arefastand easytoperform. Laser Ablation (LA) as asampling method rapid 14C/12Canalysis can be Here,wedescribe aconceptuallynew chemosensorfor spermine.2 Thesen- performed at high spatial resolution. Here, CO2 is generated by material de- sor is basedonacharge-frustrated amphiphilewith ahighlyfluorescent composition upon exposure of focused high intensity laser pulses and can head group. Analyte-induced aggregationresultsinpronounced fluores- directlybeintroducedintothe gas ionsource of an Accelerator Mass Spec- cence quenching. Thesensitivity andselectivityofour sensor is significant- trometer(AMS) [2]. For the direct coupling of LA with AMS aLAunit was ly better than whathas been describedpreviously for optical spermine developed consisting of an ablation cell (effective volumeofapproximately chemosensors. We areable to detect of spermine down to the low 0.6 mL)that combines arelatively short washouttimewith minimal particle nanomolar concentration range. Furthermore, our system displaysavery depositiononthe cellwindowand walls. Thisspecific design leads to short good selectivityoverspermidine andotherbiologicalrelevantamines. Pre- measurement times and also reducescross-contamination. Furthermore, liminary tests with artificial urine areevidence that our sensorcan be used large samples (150 x25x15mm3)can be hosted by the cell and moved by in acomplex matrix. apositioning systemathigh spatial resolution relative to the laser beam.An ArF-ExcimerLaser (λ =193 nm)isguided to the samplesurface, allowing for ablationofe.g. carbonatesatascale of less than 100 μm. Simultaneous observation of thesampleand the ablation process is possible. First results 14 on the CO2 production rate on carbonates during LA and first Cmeasure- ments will be shown. References [1] Libby, W.F., Anderson, E.C., Arnold,J.R., Science 1949,109, 227. 1. J. P. Bant et al.Clin. Nutrition, 2005,24, 184. 2. I. Hamachi et al.J.Am. Chem.Soc., 2011,133, 1670. [2] Ruff, M., Wacker, L., Gaggeler, H.W., Suter, M., Synal,H.A., This workwas supportedbythe Swiss NationalScienceFoundationand Szidat, S., Radiocarbon 2007, 49, 307. by the EPFL.

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Rapidand sensitive analysis of proteins by labeling with 3-(2- Particle Number Concentration determinedbyICP-MS furoyl)quinoline-2-carboxaldehyde (FQ), separation with CE-SDS and LIF detection SabrinaGschwind, LourdesAja Montes, DetlefGünther

Franka Kálmán, Antoine Fornage,MiriamS.Goyder ETHZurich, Wolfgang-Pauli Str.10, 8093 Zurich,Switzerland

InstituteofLife Technologies, University of Applied Sciences Western Duetotheir unique propertiesnanoparticlesare usedinmanydifferent Switzerland(HES-SO Valais), RouteduRawyl 64, 1950 Sion, Switzerland fields of application. In ordertoassess possible risks of nanoparticleson environmentand human health,theyneedtobecarefullycharacterized. Therefore, theirmost important properties(mass/size,elemental composi- Laserinduced fluorescence (LIF) detection afteranalyte derivatization with tion, morphologyand particle numberconcentration) must be described. afluorescent tag provides ultimate high detection sensitivity for proteins However, different complementarytechniqueshavetobeconsidered to ob- separated by capillary electrophoresis-sodium dodecylsulfate(CE-SDS). tain allthese information. However, oftenthe process is not quantitative and is timeconsuming, par- Recentstudies have shown that it is possible to obtainmass/size andele- ticularly as subsequent protein purification is necessary to isolatethe protein mental compositionsimultaneouslyusing amicrodroplet generationinduc- [1-3] from non reacted fluorescent reagent,asisthe case withthe wellknown tivelycoupled plasma mass spectrometer(MDG-ICP-MS) . fluorophoreTAMRA. Additionally, TAMRAhas an excitationmaximumat Thisworkfocusesonthe evaluationofparticle numberconcentrationof 550 nm,thus not providingmaximumsensitivity using the 488 nm argon silver nanoparticle suspensionswith different nanoparticle sizes(20 nm,60 ion laser commonly employed with CE-LIF [1]. nm,80nm, 100 nm)atdifferent initialconcentrations as well as different storagetimescomparing twotechniques: single particle (sp)-ICP-MS[4] and We will discuss derivatization of proteins with 3-(2-furoyl)quinoline-2- MDG-ICP-MS. carboxaldehyde (FQ) for CE-SDS-LIFanalysis. FQ reacts very quickly with primary amines of proteins to formahighlyfluorescent product that is exit- [1]Gschwind, S.;Flamigni,L.; Koch,J.; Borovinskaya,O.; Groh, S.; ed at 488 nm using the conventionalargon ion laser. Non reacted reagent Niemax,K.; Günther, D., J. Anal. At.Spectrom., 2011, 26,1166. does notfluoresce anddoesnot require removal [2]. Quantitative aspects of [2]Borovinskaya,O.; Hattendorf, B.;Tanner, M.; Gschwind, S.;Günther, the reaction are investigated and reaction conditions are optimized. Detec- D., J. Anal. At.Spectrom., 2013, 28,226. tion of recombinantproteins produced at low levels in Chinese Hamster [3]Gschwind, S.;Hagendorfer, H.;Frick,D.A.; Günther, D., submitted. Ovary cell lines in bioreactorsisdiscussed. Using FQ-labeling with CE- [4]Laborda,F.; Jimenez-Lamana,J.; Bolea, E.; Castillo,J.R., J. Anal. At. SDS-LIF at 488 nm limitsofquantification (LOQ) <10 ng/mlare achieved. Spectrom., 2011, 26,1362.

1. Oscar Salas-Solano, Brandon Tomlinson, Sarah Du, Monica Parker, Alex Strahan, Stacey Ma. Anal. Chem. 2006 78(18), 6583-6594. 2. David AMichels, Lowell JBrady, AmyGuo, Alain Balland. Anal. Chem. 2007 79(15), 5963-5971. 466 Chimia 2013, 67, Nr. 7/8 analytical sciences

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Mass Discrimination in High-Mass MALDI-MS IonmobilitySpectrometrycoupled to Laser-Induced Fluorescence

Simon Weidmann,Gediminas Mikutis, Konstantin Barylyuk, Renato Zenobi Vladimir Frankevich,Pablo Martinez-LozanoSinues, Konstan- tinBarylyuk, andRenatoZenobi ETH Zürich, Department of Chemistry and Applied Biosciences, CH-8093 Zürich, Switzerland ETH Zurich,WolfgangPauli Str. 10, 8093 Zurich,Switzerland

When complexmixtures of high-mass biomolecules such as DNA, peptides, Laser-Induced Fluorescence (LIF)isawell-knownspectroscopic method proteins, or synthetic polymers are investigated, qualitative as well as quan- which is widely appliedfor sensitiveprobing of thestructure of molecules titative information is of interest. Matrix-assisted laser desorption/ionization due to itshighspecificity to themicroenvironment, andtostructuraldetails. mass spectrometry (MALDI-MS) is one of the methods for investigating such In ordertoenableanalysisbyoptical spectroscopy,non-fluorescentbiomol- mixtures. Determination of the ratio of the individual constituents is cru- eculescan be conjugatedtofluorescenttags, which aresmall moleculeswith specifiedoptical properties.BothLIF spectroscopyand Differential mobili- cial. Previous studies showed that mass bias is partially caused by the mi- ty analyzers(DMAs)havebeen extensivelyusedfor characterizationof crochannel plate (MCP) detectors used [1]. However, recently we have shown different compounds, butusually separately. that high-mass molecules do not yield the same response overthe whole m/z We report on an improved design of DMAcoupled with LIF, to simultane- range when special high-mass detectors, such as ion conversion dynode (ICD) ouslyretrievetwo-dimensionalinformation on theelectricalmobilityand detectors, are used [2]. In this work the relative response for proteins with fluorescence spectroscopyofgas-phase ions.Thisenhanced design includes molecular weights from 35.9–129.9 kDa are determined for awide range of an ionfunnelinterface at theinput orificeofthe DMAand anozzlebeam molar ratios and factors influencing the response are discussed. stageatthe output of theDMA.These improvements allowdetectingfluo- We found that the mass discrimination is less pronounced when using an ICD rescence notonlyfrompuredyesand theirclusters, as wasdemonstrated detector,compared to the use of conventional MCP detectors. The response recently, butalsofromfluorophor-taggedbiomolecules. Complexmixtures wasfound to depend on the laser power used for MALDI; low-mass ions of fluorescentcompounds canbeseparated by theDMA andstudied by LIF. are discriminated against with higher laser power.The effect of mutual ion AFRETexperiment with aBODIPY-cRh6G complexdemonstratesthe new suppression as afunction of the proteins used and their molar ratio is also capabilitiesofthe instrument.Thisunique instrument combinationalsopro- shown: mixtures consisting of the same proteins that only differ in mass show videsapowerful platform forprobing fluorescentproteinsinthe gasphase. Green fluorescentprotein (GFP)was tested on anew setup. In contrast to less discrimination than mixtures consisting of different proteins with similar thehighvacuum,where no GFPfluorescence hasbeendetected,the pres- masses. Furthermore, mass discrimination dramatically increases for molar ence of aLIF signal at theoutputofDMA couldexplain some specific fluo- ratios farfrom 1. Finally,wepresent clear guidelines to choose the experi- rescentpropertiesofGFP in thegas phase. Giventhatbothconformation mental parameters such that the measured response matches the actual molar andfluorescenceare keypropertiesofbiologicalmoleculesinthe gasphase, fraction as closely as possible. we expect that ourenhanced design will answer thequestionwhether or not [1] T. B. Farmer,R.M.Caprioli, J. Mass Spectrom 1995, 30,1245. gas-phaseproteinsretaintheir liquid-phasenativestructure. [2] S. Weidmann, K. Barylyuk, N. Nespovitaya, S. Mädler,R.Zenobi, Anal. Chem. 2013, 85,3425.

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In-silicoscreeningfor metaboliteinterferencesinquantitative CombiningDemultiplexingand Label-free Quantification forHigh- LC-SRM/MS resolution Data-independent Acquisition LC-MS/MS Analyses

TobiasBruderer, Emmanuel Varesio, Gérard Hopfgartner Aivett Bilbao1,2,Ying Zhang1,Dario Bottinelli1,BandarAlghanem1, Frédéric Nikitin2,JeremyLuban3,CaterinaStrambio De Castillia3,Markus Life Sciences Mass Spectrometry,School of Pharmaceutical Sciences,Uni- Mueller2,Frédérique Lisacek2,EmmanuelVaresio1,GérardHopfgartner1 versityofGeneva, University of Lausanne,QuaiErnest-Ansermet 30, 1211 Geneva,Switzerland 1. Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences, UniversityofGeneva, UniversityofLausanne. QuaiErnest-Ansermet The validationofLC-SRM/MSmethods in quantitativebioanalysis requires 24, CH-1211 Geneva,Switzerland. ascreeningfor potentialinterferences causedbycoelutingcomedications or 2. Proteome Informatics Group, Swiss InstituteofBioinformatics.Rue theirmetabolites. The limitations of currentapproaches have been addressed Michel Servet1,CH-1211Geneva, Switzerland. by apreviouslypublishedscreeningstrategyfor comedications basedon 3. UniversityofMassachusetts, Medical School, PrograminMolecular predictedLCretentiontimesand MS precursor interferences[1]. Medicine.Plantation St 373, MA 01655 Worcester,USA.

The screening strategyhas been extendedfromonlycomedications to in- There areseveral advantagesofdata-independentacquisition (DIA)over clude theirmost likelymetabolites. The elutionorder of hydroxilatedvera- data-dependent acquisition (DDA)schemes for proteomicanalysesofcom- pamil metabolite isomerscouldbepredictedcorrectly basedontheir logD plex peptide mixtures(e.g. no bias toward high abundant peptidesand re- values if thenew consensus algorithmofACD Labs 2012 wasapplied. producibility). One of theseDIA strategies is SWATH,where aresolving Q1 window (e.g.20-30u) is steppedrepeatedlyacross amass rangeand Retentiontimeswere predictedbased on amultiple linear regression be- high-resolution MS/MSspectra aregenerated forall precursor ions within tween measured retentiontimesfor aset of 25 standards, thelog Dand ad- the Q1 window (multiplexed spectra). ditionalmoleculardescriptors. Good accuracy hasbeen achieved. The setof Whilethereare available toolstoquantify SWATHdatainatargeted man- standardswill be extendedtoseveral hundred compounds andits accuracy ner[1],theyare limited toolsfor simplifyingMS/MS spectra (demultiplex- investigated in detail with theleaveone out (LOO)approach. ing)including the corresponding precursor ions with highmass accuracy that can be effectivelyusedfor peptide sequence identificationaswellas [1]Tobias Bruderer, EmmanuelVaresio,Serge Winter andGérard simultaneous label-free quantification, whichstatesthe objective of this Hopfgartner, Bioanalysis 2012, 4(15),1907-1917. work. The currentefforts areconcentratedtodevelop amodel for precursor ion assignment basedinthe identificationand correlationbetween the residual precursor ion in the MS/MSspectra andthe precursor ion in the MS spectra addedatthe beginning of each acquisition cycle.

[1]L.Gillet,P.Navarro,S.Tate, H. Röst, N. Selevsek, L. Reiter,R. Bonner, R. Aebersold., MolCellProteomics. 2012, 11,111. analytical sciences Chimia 2013, 67, Nr. 7/8 467

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Selectivity andsensitivity evaluation in LC-SRM-based peptidequanti- MALDI Microarrays for fication by usingMRM3ordifferential ion mobility spectrometryap- rapid absolute quantification and single-cell analysis proaches Stephan Fagerer, Martin Pabst, Thomas Schmid, Alfredo Ibáñez, Bandar Alghanem1:AivettBilbao1,3;YingZhang1;Dario Bottinelli2; Robert Steinhoff, Jasmin Krismer and Renato Zenobi Frédéric Nikitin3;Markus Mueller3;Frédérique Lisacek3;JeremyLuban2; Caterina Strambio De Castillia2 ;Emmanuel Varesio1;GérardHopfgartner1 ETH Zürich, Wolfgang-Paulistr. 10, 8093 Zürich, Switzerland

1 Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences, We present anew sample application method for matrix-assisted laser de- UniversityofGeneva, UniversityofLausanne,Geneva, Switzerland sorption/ionization (MALDI). It is comprised of amicroarray chip made of 2 UniversityofMassachusetts,Medical School,Program in MolecularMed- either glass or metal that contains hydrophilic spots with 100 or 200 µm icine, Worcester, MA,USA diameter in an hydrophobic coating created by laser machining of apolysi- 3 Proteome InformaticsGroup,Swiss InstituteofBioinformatics, Geneva, lazane coating. Nanoliter volumes can be reproducibly aliquoted by moving Switzerland aliquid sample across the array. Single cells can be isolated by dragging a cell suspension across the array with astatistical number of single cells stay- Selected reactionmonitoring(SRM) in conjunctionwithstableisotope dilu- ing behind hydrophilic spots. After application of aMALDImatrix, the tion hasbeenemployedasastandard workflow forpeptidesquantification sample can be analyzed. in mass spectrometry. Generallytoassess theaccuracyofthe measurement severaltransitions arerecorded which increasesthe complexity anddecrease We have successfully applied this system for the detection of primary me- thesensitivity of theassay.Thisisparticularly critical forthe quantification tabolites (e.g. phosphoenolpyruvate, adenosine triphosphate and uridine of lowabundantproteinsincomplex biologicalsamples. diphosphate glucose) from various unicellular organisms such as S. Cerevesiae (Baker's yeast) and different algal species. When using trans- This studyattemptstoevaluatethe pros andconsregarding selectivityand parent targets we were able to measure the same single algal cells of H. Plu- sensitivityofalternative approaches.Inparticular, multiple reactions moni- vialis cells both with Raman microscopy and MALDI mass spectrometry. toring cubed(MRM³)and differentialion mobilitymassspectrometry (DMS)wereevaluated forquantitativepeptidesLC-MS analysis in Buffy Recently, we have also started to apply the self-aliquoting targets for quanti- coatsproteinsextracts. Theselectivityand/orsensitivity were assessed tative MALDI analysis. Two distinct advantages exist with our target. First, basedondifferent aspectssuchaspeptide signal,matrixbackground level, inhomogeneous matrix/analyte crystallization is not of consequence since iontransmission loss, andresolving powerseparation. the small spot size allows for the ablation of the whole reservoir. Second, a high number of repeats (10-20 per spot) that are possible with each sample deposition within seconds increase the statistical relevance and reliability. Calibration curves for apeptide mix showed coefficients of variation of 0.98 or better without internal standard. This is remarkable since MALDI is usu- ally not used for quantitation due to high spatial signal fluctuation.

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Increasingpeptideidentification by mass spectrometry-friendly sample Determination of bindingconstants of aptamer-ligand complexes using preparation andenhanced LC-MS/MS acquisition electrospray ionization mass spectrometry

Ying Zhang1,Dario Bottinelli1,Aivett Bilbao1,2,BandarAlghanem1,Frédé- Basri Gülbakan, Konstantin Barylyuk, Renato Zenobi ric Nikitin2,Markus Müller2,Frédérique Lisacek2,JeremyLuban3,Caterina StrambioDeCastillia3,EmmanuelVaresio1,GérardHopfgartner1 Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland 1. UniversityofGeneva, Life Sciences Mass Spectrometry, Boulevard d'Yvoy20, 1211, Geneva,Switzerland. The study of the interaction of different ligands with nucleic acids is an ex- 2. Swiss InstituteofBioinformatics,Proteome InformaticsGroup, citing area in modern . Anew class of molecular affinity RueMichel-Servet1,1211, Geneva,Switzerland. probes called “aptamers” has gained great importance recently. Aptamers 3. UniversityofMassachusetts,Medical School, PrograminMolecular are single-stranded oligonucleotides,which can bind to other molecules Medicine,373 Plantation St,01605, Worcester,MA, USA. with very high affinity and specificity. Despite the great promise of ap- tamers, very few studies exists on aptamer-ligand interactions. Most of them In proteomics,LC-MS/MS analysis of peptidesisgenerally performed using were carried out by optical spectroscopy techniques. Development of label- adata-dependent acquisition (DDA)approach to determine structuralinfor- free MS methods for studying aptamer-ligand interactions will therefore be mation basedonthe peptide precursor ion selected for fragmentation. How- of great value. In this study we use ESI-MS and MALDI-MS to investigate ever,the nature of thisMS/MS spectra acquisition creates asampling bias aptamer-small ligand and aptamer protein interactions. ESI-MS against those relativelylessabundantpeptides in complex matrices. experiments were conducted with an adenosine aptamer. ESI mass spectra of only aptamer solutions resulted in ions at m/z 1413 and 1696, correspond- In ordertoyield ahighproteome coverage andsamplethroughput forana- ing to the −6 and −5 charge states. Upon addition of varying concentrations lyzing human primary monocytederiveddendritic cells (MDDCs) protein of adenosine, 1: 1and 1: 2aptamer/ligand complexes at m/z 1750 and 1803 extracts, we optimizedand evaluatedaMS-friendlysamplepreparation pro- were observed. We have found that optimal instrumental conditions to ob- cedurewhichresulted in amore than 10% increaseofidentified proteins. serve adenosine aptamer and and adenosine complexes were: cone voltage, The performanceofthree DDAMS/MS acquisition approaches,namely: i) 0.8 kV; sampling cone voltage 30 kV, source offset 30 kV. The concentra- generaltechnical replicates acquisition; ii) enhanced acquisition with exclu- tion of ammonium acetate buffer was found to be very critical to maintain sion/inclusion lists; andiii)technicalreplicates acquisition with extended aptamer-ligand interactions in nondenaturing conditions by ESI with 50 mM LC separation, were also compared with the common aimtoimprove the NH4Ac being the optimum value. Upon increasing the NH4Ac concentration number of peptide identification in MS analysis. from 50 mM to 100 mM, we have seen adecreasing trend in complex for- mation where no complex is seen at 100 mM. Addition of organic solvents helped to improve signal intensities of aptamer/ligand complexes. The dis- sociation constants (Kd)measured via nanoESI-MSwere compared with solution phase (Kd) values. 468 Chimia 2013, 67, Nr. 7/8 analytical sciences

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DifferentiatingBreast Cancer Cellsvs. NormalMammary Cellsbased Binding of cancer drugs to higher-order nucleic acids on theirvolatilesignature by Secondary ElectrosprayIonization Mass Spectrometry Yvonne Hari, Silvan R. Stucki, Stefan Schürch

Jingjing He1,3,Xue Li1,Maija Hollmen2,Pablo M-LSinues1,Michael Departement Chemie und Biochemie, Univeristät Bern, Freiestrasse 3, 3012 Detmar2,RenatoZenobi1* Bern, Switzerland

1. DepartmentofChemistry andApplied Biosciences,ETH Zurich, CH-8093 Zurich,Switzerland Higher-order nucleic acid structures like DNA quadruplexes have lately 2. InstituteofPharmaceutical Sciences,ETH Zurich,CH-8093 Zurich, attracted increasing attention due to their proposed biological functions. Switzerland DNA quadruplexes are involved in various processes of cell cycle control, 3. State KeyLaboratoryofBioactiveSubstance andFunction of Natural including the regulation of telomere length and gene expression. These Medicines, InstituteofMateria Medica, ChineseAcademy of Medical Sci- regulatory properties render DNA quadruplexes apromising target for or- ences andPekingUnion Medical College, Beijing100050, P.R. China ganic and organometallicanti-tumor drugs.

Recently, there hasbeen increasingevidence for arelationship between vol- Cisplatin is the cornerstone of modern cancer therapy. Its cytotoxicity is atilecompounds released by human the bodyand cancers. Differentiating mostly attributed to the binding of adjacent guanine nucleobases, which malignancythrough itsvolatilemetabolicsignaturealong with identification results in 1,2-intrastand crosslinks in cellular DNA. Cisplatin was shown to of such volatilemetaboliccompounds mayprovide potential biomarkers for influence the gas-phase dissociation of single- and double-stranded DNA as early andnoninvasive diagnosisofmalignant diseases [1]. well as quadruplex DNA. The precise binding sites of cisplatin were deter- We analyzed the volatilemetabolicsignatureofthreetypes of human breast mined by the means of high-resolution tandem mass spectrometry [1,2]. cancercelllines (T47D, SKBR-3, andMDA-MB-231) versus normal human mammarycells (HMLE).Volatile compounds in the headspaceofcondi- However, due to severe side effects of cisplatin, alternative organometallic tionedculturemedium were directly fingerprintedbysecondary electrospray compounds, including phenanthroline derivatives, ruthium-arene-complexes ionization-mass spectrometry. The mass spectra were subsequentlytreated and multinuclearplatinum compounds,are tested for future therapeutic ap- statistically to identify discriminating features between normalvs. cancerous plication. The goal of this project is to elucidate the interaction of these celltypes. drugs with higher-order DNA structures, to reveal the corresponding bind- Differentsamples were classifiedbyusingfeature selection, followedby ing patterns, and to localize specific binding sites. Latest results on the DNA principalcomponentanalysis (PCA).Inaddition, high-resolution mass binding properties of (1,10-phenanthroline)-platinum(II) are presented. spectrometryand fragmentation of the most discriminantmolecules cangive some clue to their chemical structure. Our studysupports the hypothesis that cancerous cellsrelease acharacteristic [1] A. Nyakas, M. Eymann, and S. Schürch, J. Am. Soc. Mass. Spectrom. odorous signaturethatmay be used as disease markers. 2009, 20,792-804. [1]Catherine Oakman et al., Int JBiochemCellBiol, 2011,43,1010– 1020. [2] S. R. Stucki, A. Nyakas, and S. Schürch, J. Mass. Spectrom. 2011, 46, 1288-1297.

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Lipidomic Profiling of Algal Populations with Single Cell Resolution Analysis of nucleotide ratios from cellular metabolism using negative Krismer Jasmin, Steinhoff Robert, Fagerer Stephan,Ibañez mode MALDI-MS Afredo, Pabst Martin, Zenobi Renato Steinhoff R.F., Krismer J., FagererS., Ibanez A.J., Pabst M., Zenobi R. ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland Studying microbial populations at the single cell level is essential for under- standing heterogeneity in biological systems. Their investigation can reveal awhole new level of information and improve our understanding of how Single cell analysis (SCA) is becoming atechnology more and more used in microbial communities work [1]. We are currently developing anew ap- medical and industrial biotechnology. [1] Important areas such as diagnos- proach for lipidomic profiling of microbial populations using ahigh-density tics or cancer therapies increasingly call for SCA tools. Our mass spectro- microarray for matrix-assisted laser-desorption ionization –mass spec- metric approach for SCA, which was first demonstrated in 2008, gives in- trometry (MALDI-MS). Chlamydomonas reinhardtii cells are dispersed on sights on the metabolomics level. [2] achip consisting of aself-aliquoting microarray. Confocal fluorescence We are interested in characterizing the energy charge in single cells and scanning locates the autofluorescentcells in the array. Thereby the fluores- furthermore want to provide adirect and simple access to individual cellular cence of each individual cell can be quantified. The MALDI-matrix is ap- responses. The use of matrix-assisted laser desorption ionization (MALDI) plied to the array using anon-contact spotting device. Using DHB in posi- as aionization method on our custom designed microarray allows high sen- tive ion mode, high-resolution MALDI-FT-ICR measurements make it pos- sitive detection of the nucleotides in single mammaliancells. We investigate sible to detect most of the lipid classes contained in the algal membranes. the use of 9-aminoacridine (9-AA)asamatrix in MALDI MS to character- Furthermore the photosynthetic pigments that are detected both by fluores- ize the ADP/ATP ratio in abiological system. 9-AA has few matrix peaks in cence microscopy and mass spectrometry can be used to cross-validate the the low mass region and is very sensitive to nucleotides(attomolar concen- two methods. The system developed shows the potential of MALDI-MS for trations). However 9-AA tends to initiate the in-source decay of nucleotides high-throughput screening of populations. to fragmentation products. The in-source fragmentation yield is influenced by general instrument settings (e.g. laser fluence, delayed extraction time). [1] Stanislav S. Rubakhin, Eric J. Lanni, Jonathan V. Sweedler, Progress toward single cell metabolomics, Curr. Op. Biotech., 2013,24,1,95-104 References [1] Fritzsch, F.S.O.,Dusny, C., Frick, O., Schmid, A. Annu. Rev. Chem. Biomol. Eng. 2012,3,129-55 [2] Amantonico, A., Oh, J.Y., Sobek, J., Heinemann, M., Zenobi, R. Ange- wandte Chemie 2008,120, 5462-5465 analytical sciences Chimia 2013, 67, Nr. 7/8 469

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Thin-film materials as reference samples for Tip-Enhanced Raman Rapidand efficient large scale isolation strategyonMPLC-ELSD-UV Spectroscopy usinggradient transferand enhanced pressure andtemperature control

Bruno Stephanidis, Lothar Opilik, Renato Zenobi ChallalS., Queiroz E.F., Kloeti W, Guillarme D., WolfenderJ.-L.

ETH Zurich, Department of Chemistry and Applied Biosciences School of Pharmaceutical Sciences,EPGL, UniversityofGeneva, Universi- Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland ty of Lausanne, QuaiAnsermet30, 1211 Geneva,Switzerland

Tip-enhanced (TERS) is avery promising technique In naturalproduct(NP)research, theisolationofbiomarkers or bioactive combining the electromagnetic enhancement of the Raman signal by afull compounds from complex naturalextract is an essentialstep for de novo metal or ametal-coated tip and the high spatial resolution of Scanning Probe identificationand bioactivity assessment.Whenpure NPshavetobeob- Microscopy.Ithas the potential to become an irreplaceable tool for na- tainedinmilligram quantities,the chromatographicsteps aregenerally labo- noscale spectroscopic analysis. However, TERS still has limitations: one of rious andtimeconsuming. An efficient wayconsistsinthe transferofopti- them is the the lack of reproducibility of the enhancement and of the spatial misedreversedphase gradient profiling conditions from analytical to semi- resolution of tips. preparativeHPLC[1].Inorder to increasethe sampleloading,Medium To overcome these limitations, there is astrong need for areference sample Pressure LC (MPLC)represents an interesting alternative since gramsof allowing for afacile testingand comparison of tips.The idealreference crude extract can be separated in onlyone step. The MPLC separationsare sample has to be agood Raman scatterer, chemicallystable and mechanical- howeverlimited by the flow rate andpressureallowedonthe glass column ly resistant. We present here our first results on inorganic thin films such as andtypical separation arecarriedout overseveral days. diamond membranes fabricated by CVD and thin films of zinc oxide depos- To overcome thisproblem, we have developed models for accurate gradient ited by ALD, which are promising reference samples. transferfromthe HPLC to the MPLC scaleand precise prediction of the separation of the extractsatthe gram scale. Adry loadingstrategyadapted to most plant extractsthatminimise pressure drop hasbeen optimised. The speedofseparationhas been enhanced by maximising the flow rate at room temperatureusing anew wayofcontrollingpressure at the columninlet. Furthermore,the pressure drop wasminimised by designingadedicated columnovenfor preparativeseparationand performseparations at up to 60°C. The improvementsofseparationobtainedare illustrated with the separation of threecrude plant extracts, each containing different classesofmolecules. In these differentcases,amajorityofpureNPs could be obtainedinmg amountsinabout one to two days. [1]Glauser G, Guillarme D, GrataE,Boccard J, Thiocone A, Carrupt P-A, VeutheyJ-L,Rudaz S, WolfenderJ-L, JChrom A 2008,1180, 90

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Laserinducedbreakdown detection(LIBD)usedasasensitivenano- LaserAblation Mass Spectrometry with Ion Funnelfor TraceElement particle detector to derive numbe rbased size distribution afte rFlow Analysis in Solids. FieldFlowFractionation (AF4) Saurabh Abbi1,Victor Varentsov2,Rolf Dietiker1,Tatiana Egorova1,Bodo a b b a Fedotova Nataliya ,KägiRalf ,SinnetBrian ,KochJoachim , Hattendorf1 andDetlefGünther1 GüntherDetle fa a 1ETH Zürich, Laboratoryfor ,Wolfgang Pauli Str. 10 LaboratoryofInorganic Chemistry, ETHZurich, 8093 Zurich 8093 Zurich,Switzerland bParticle Laboratory, Swis sFederal InstituteofAquatic Scienceand Tech- 2FairGmbH, Planckstraße 1, 64291 Darmstadt, Germany nolo gy,CH-8600 Dübendorf Laserablation mass spectrometry(LAMS)for elemental analysis of solids Nanopartic lesare omnipresentinour day-to-day life. However, ourknow- hasbeen introduced severaldecades ago. Theproblems causedbymatrix ledgeabout theirimpactand behavior is still insufficientand analytical dependent ion yields howeverput aseverelimitation beforethe broader techniques able to mo nitorthatare of need.Onlyin2011, theEuropean acceptanceofthistechnique.Anotherlimitation of LAMS is the hugeener- Commission issued an official recommendatio nonadefinition of nanopar- gy spread of theions generated by laser ablation in vacuum. ticles [1]. As aconsequence of theissueddefinitio n, techniques that are In order to minimizethe ion spread,Wangetal. usedHeliumatmoderate able to detect thenumbersizedis tributio nwill become of greatinterest. pressure as buffergas to reducethe kinetic energyofions to improve the In ourworkweusedacomb inationofLIBDand AF4todeterminethe sensitivity especiallyfor lighter ions whenusing electrostatic ion optics [1]. numberbased size distribution of polystyrenenanoparticlesinliquidsus- Anotherapproach to thermalizeions from laserablation is investigated in pensions.Asafirststep, theexperimentsonthe separation of mixture of thisstudy. An ion funneloperatedatapressure between 0.1 and10mbar is nanoparticleswereperformed in abatch mode.Thisallowedustotestfor inserted between the ablation siteand the electrostatic ion optics of atimeof theabsence of aggregated nanoparticle sinthe AF4channel by determining flight mass spectrometer(TOFMS).Incontrasttoprevious configurations with LIBD sizes of nanopartic les. Afterwards themonodisperseand poly- [2], thision funneldoesnot require axial accelerationofthe ionsdue to the disperse suspensionswereanalyzedonline. TheLIBDsystemwas cali- specific gasflow dynamics inside the funnelitself. After thefunnel ions brated basedonthe energy curves (ors-curves-thedependenceofthe exhibitalow kinetic energy spread andare moreefficientlyfocused to the breakdownprobabilityonthe laser pulseenergy) recorded fordifferent sizes entranceapertureofthe TOFMS. Thissimplifiesthe construction andopera- andconcentrationsofpolystyrene nanoparticles. Then as theresults of the tion of the funnelsignificantly. LIBD andAF4 couplingfractograms were obtained.Based on theinforma- First resultsinvestigating the dependence of ion transmission on operating tion fromLIBDand AF4measurements we developedanalgorithm con- parameters of the ion funnel as Rf-frequencyand amplitude as well as buffer vertingthetime resolved breakdown probabilitiesintoparticleconcentra- gaspressurewill be discussed in thispresentation. tions. With thesodevelopedAF4-LIBDsystem, size andnumberparticle concentratio nofpolystyrene nanopartic lescould be determined,thatmeet 1. Huang, et al., Mass Spec. Reviews, 2011. 30(6) therequireme ntsofthe definition of nanoparticlesproposed by EU.Further investigationwillfocus on theother typesofnanopartic les. 2. Shaffer, S.A., et al., Rapid Comm.inMass Spec., 1997. 11(16). [1]Recommendation[2011/696/EU] 470 Chimia 2013, 67, Nr. 7/8 analytical sciences

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Middle-DownProteomicAnalysis of Embryonic Proteins Secreted Detection of Aptamer-Protein Complexesusing MALDI-MS During the IVF Procedure Fan Chen, Basri Gülbakan, RenatoZenobi ÜnigeA.Laskay,1 KristinaSrzentić,1 Tanja Panić-Janković,2 Michel Monod,3 Goran Mitulović,2 YuryO.Tsybin1 Department of Chemistry and Applied Biosciences, ETH 1Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne,Switzerland, Zürich, CH-8093 Zürich, Switzerland 2Medical UniversityofVienna,1090 Vienna,Austria, 3Centre HospitalierUniversitaire Vaudois,1015 Lausanne,Switzerland Aptamers are single stranded DNA or RNA oligonucleotides, which fold Mass spectrometryisapowerful techniquecapable of identifying thousands into unique three-dimensional shapes and form abinding pocket or alloster- of proteins from complex biologicalsamples.Inbottom-upproteomics the ic site to accommodate their target molecules with high affinity and speci- proteins aredigested in short, 6-20 residue long peptides, which areeasier to ficity. Although aptamers have been applied to achieve fast and sensitive separate andanalyze.However, the technique is not idealbecause of the detection of proteins, understanding the non-covalent interactions in protein- very high numberofpeptidesgenerated that oftenhaveuninformative se- aptamer complexes by mass spectrometry is still alargely untouched field. quences.Asopposedtobottom-up, by producing mid-range (30-70 resi- Here, we presented the first report on protein-aptamer interactions by ma- dues) peptides, the samplecomplexityisdecreasedand analysis of asingle trix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) . peptide can offerextensive sequenceinformation andreliable protein identi- We investigated the effect of MALDI matrix, sample preparation method, fication. Although the need for thispeptide sizerange hasbeenrecognized, laser energy and concentration of aptamer and proteins on the specific non- the reproducibleand facile production of these peptidesisnot trivial.We covalent complex detection.Afteraseries of optimizations, 6-aza-2- have comprehensivelycharacterized the aspartic protease Sap9, andfound thiothymine (ATT)and amodified sandwich sample preparation method that it is active in abroad pH range andmaintains itsactivityattemperatures provided optimum detection efficiency. By using optimized conditions, we 15-45 °C.Peptideswereseparated on anano-LC C8 columnand analyzed were able to directly observe the complexes of thrombin and thrombin bind- with astate-of-the-artOrbitrapEliteETD FTMS.43out of the 48 proteins ing aptamer (TBA). We also confirmed that the complex observation is due in UPS1commercial standard were identified. Thesameexperimental con- to specific non-covalent interactions, rather than non-specific clusters ditions were applied to theanalysis of secreted embryonic proteins from the formed in the MALDI plume. We further confirmed the capability of culturemedium. Althoughtheseproteins areexpected to carry multiple MALDI-MS to study protein-aptamer interactions by using lysozyme and its phosphorylation sites,typicalbottom up proteomics identifiedonlyahand- corresponding aptamer. The complex stability in MALDI strongly depends ful of proteins with low sequencecoverage. Sap9 digestion resulted in en- on the dissociation constant of the complexes in solution phase: the stronger hanced sequencecoverageofthe embryonic proteins andahighernumber thrombin-TBA29 complexes was observed to yield alarger fraction of sig- of total proteins identified.Inaddition, severalphosphorylation sites were nal at the mass of the intact complex than the weaker thrombin-TBA15 reliablyassigned.Therefore, we have developed aproteolytic method for complex. This indicates that the non-covalent interaction strength in solu- protein characterizationtargeting mid-sizepeptides, andsuccessfullyap- tion is reflected in the MALDI mass spectra. plied it to solve aclinicalresearchquestion.

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Low-volume sampling directly coupled to ESI-MS analysisusing a Influence of wetgas atmosphere duringablation of zircon CapillaryGap Sampler on U/Pb ratios andresultingagesusingLA-ICP-MS

VolkerNeu1,Roger Steiner2,StephanMüller2,Christof Fattinger2, SteffenAllner,LucaFlamigni, Daniel Tabersky, Joachim Koch, Detlef Renato Zenobi1 Günther

1 DepartmentofChemistryand Applied Biosciences,ETH Zurich, CH-8093 LaboratoryofInorganic Chemistry, ETH Zurich, Zurich,Switzerland. Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland 2 F. Hoffmann-LaRoche AG,pRED, Pharma Research &Early Development, Discovery Technologies, Grenzacherstrasse 124, CH-4070 U/Pb-basedgeochronology is one of the most frequentlyapplied dating Basel, Switzerland. methods in the research field of geology for samples older than about 100 million yearsuptothe oldest samples of more than 4billion years. Zircon is Chip-based microfluidic devicescoupled to ESI-MS have been commercial- the preferredmineral for thisdatingmethod, because Uisincorporated in ly available for severalyears, but still have limitations with direct introduc- the crystallattice during crystallization,whereas Pb is mainly rejected. This tion of samplevolumes <1µL as well as sitespecificityinsamplepickup, leads ideallytothe absenceofinitial leadand allows dating singlegrains. whichisboth of highinterestfor biomedical applications. We present a Laserablationinductively coupled plasma mass spectrometry (LA-ICP-MS) “CapillaryGap Sampler”asanew, non-chip basedmicrofluidic platform, is afastand sensitive method for solidsampleanalysisand pairs well with whichallows for robust andreproduciblesampling of injection volumes of the need for analysisoflarge sets of zircons in geological sedimentstudies fewnanoliterscombinedwith direct ESI-MS analysis. [1]. The core of thesampler consists of aliquid bridge of severalnLformed However,the LA of zircons causes fractionation of Uand Pb resulting in an withinaµm-sized gapoftwo capillaries. The capillaries areenclosed within unstable U/Pbratio overthe time of ablation [2]. This processisnot fully an air-tightchamber,whichisheldunderaregulated overpressureofseveral understood yet, but aheat-inducedphase separation with enrichment of Uin millibars. Oneofthe capillaries consists of stainless steeland actsdirectly the wall of the ablation pit[3] seemstobeamajor cause. as the ESI-MS interface. Asolidpin is used for sampleuptake anddelivery. In combination with the liquidbridge as an “open” introduction system, this To reducethiseffect,coolingand faster transfer of heat from theablationpit designminimizes sampleadsorption/sticking on surfaces duringinfusion. can be achievedbyapplying wetablation, i.e. underwater ablation or abla- The system hasaninternalvolumeofabout 120 nL,whichisappropriatefor tion with wetcarrier gas. Therefore,the LA processwas studied with re- samplevolumes of 10-12nL, limiting sample dilution to aminimum.Fur- specttodifferent degreesofhumidityinthe carrier gasand the influenceon ther system characteristics areminimal peak widths of about 6s,sub- the crater morphology andthe resulting U/PbratiosmeasuredbyICP-MS femtomoledetectionlimits, andtotal injection cycletimes <12sincluding will be presented. pin washing anddrying. Applications on peptide characterizationaftersolid phase synthesis using single beadsand bioassaymonitoring demonstrate the [1] Kosleretal., Chem.Geol., 2002, 182,605 potential of thisnew sampling tool for afastand sequential analysis of sam- [2]Kuhn et al., JAnal. At. Spectrom. 2010, 25,21 ples whichcontain multiple analytes andare of limited sample amount. [3]Kosleretal., JAnal. At.Spectrom. 2005, 20,402 analytical sciences Chimia 2013, 67, Nr. 7/8 471

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Analysis of the mechanism of NCO- loss and the consecutiverelease of a Capillary zone electrophoresis appliedtothe analysisofmicro- - PO3 groupfrom highly charged oligonucleotides heterogeneityintherapeutic monoclonal antibodies

Eberle Rahel, Nyakas Adrien, Stucki Silvan R., Schürch Stefan Anne-Laure Gassner,SergeRudazand JulieSchappler

Department of Chemistryand , University of Bern, Frei- School of Pharmaceutical Sciences,UniversityofGeneva,Universityof estrasse 3, CH-3012 Bern, Switzerland Lausanne, Bd d’Yvoy20, 1211 Geneva 4,Switzerland

Mass spectrometry is the method of choice to analyze modified oligonucleo- The developmentofmonoclonal antibody(mAb) pharmaceuticalshas been tides. The elucidation of gas-phase dissociationmechanismsofmodified constantlygrowingsincethe introduction of the first therapeuticmAb in and unmodified nucleic acids is of utmost importance to facilitate rapid and 1986. However,astheyexhibitahighmolecular complexity, small changes potentially automatedsequence analysis of artificial DNA and RNA. In this in production conditions, storage or exposure to lightorchemicalsare study the fragmentationbehavior of highly charged oligodeoxynucleotides sources of micro-heterogeneity, possiblyaffecting antibodycharge, sizeor (ODN) was investigated.Itwas found that the fragmentation of ODNs ex- glycosylation pattern. As thesemodifications mayalter the therapeuticac- hibiting ahigh charge level (ratio of theactual to the total possible charge) tivityand induce side effects, analytical methods capable of evaluatingmi- differssignificantlyfrom those with alow or medium charge level by fol- cro-heterogeneityare essential [1]. lowing so far unreported, highly selective dissociation channels. Presence of Capillaryelectrophoresis is particularlywellsuited for mAb analysis thanks aterminalthymine (3' or 5') results in anew fragmentation pathway involv- to itshighresolution andlow sampleconsumption. Capillaryzone electro- ingabstraction of acyanate anion (NCO-). Thisprocess frequentlycoincides phoresis (CZE)providesseparationselectivitybased on charge andsize, and withthe loss of water from the 3'-endorofCH2Ofrom the5'-end. Addi- mightcomplement capillary gelelectrophoresis (size) andcapillaryisoelec- - tionally, aconsecutive release of aPO3-ion was observed for most ODNs. tric focusing(charge) that areroutinely used in industry. Amajor issue in - Introduction of aphosphorothioate group allowed to pinpoint the PO3 loss the analysis of mAbs by CZEconcerns adsorptionofthese biomolecules to to the terminal phosphategroup.Furthermore, experimentswith methyl- the capillarywalls,leading to poor resolution and recovery. phosphonate-modified ODNs wereperformed to block the phosphate- In thiswork, various static capillarycoatingsand background electrolytes oxygenfor participating in the dissociation mechanism. To elucidate the were investigated to find the optimal conditions associated with good reso- impact of theribosesugar moiety on the NCO- loss, thegas-phase dissocia- lution between isoforms, minimized adsorption, andacceptable repeatability tion of highly charged homo-DNA was investigated. Since all homo-DNA for two mAbs.Tomonitorthe adsorptionofcommercial intact mAbs, four sequences exhibited terminal cyanateloss, theabstractionofNCO- is likely criteria,namelypeak efficiency, migrationtimerepeatability,EOF change, to be sugar-independent. However release of waterfrom the4'-end or of andprotein recovery,wereevaluated. Finally, the repeatability andinterme- CH2Ofrom the 6'-end was not observed and furthermore no proof for sub- diate precision obtainedwith the selected operating conditions were as- - sequent abstraction of the PO3 -ion was obtained. sessed from the mAbpatterns andapplied to othercommercial mAbs.

[1]FeketeS.etal. Trends Anal. Chem 2013, 42,74

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Analysis of proteintherapeuticsbycapillary zone electrophoresis Traceelementquantification in ancient copperartifacts by portable coupled to mass spectrometry laser ablation sampling andsubsequent LA-ICP-MSanalysis

Anne-Laure Gassner, Sadegh Motamedi, JulieSchappler andSergeRudaz Marcel Burger,RetoGlaus, Vera Hubert, Samuel vanWilligen, Marie Wörle-Soares, Detlef Günther School of Pharmaceutical Sciences,UniversityofGeneva,Universityof Lausanne, Bd d’Yvoy20, 1211 Geneva 4,Switzerland ETH Zürich, LaboratoryofInorganic Chemistry, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich The developmentofprotein therapeutics is expanding rapidlysince the ad- vent of biotechnology. However, these molecules areparticularlyfragile and Laserablationinductively coupled plasma mass spectrometry(LA-ICP-MS) their production andstoragemust be strictlycontrolled.Despiteall precau- is averyversatile andpowerful technique for elementaland isotopic analy- tions,these proteins can be degraded anditisessential to check the quality sis of solidsamples in aquasi-nondestructive manner. It allows for quantifi- of the producttoavoid anyside effectstothe patient [1]. Therefore, awide cationofmajor, minor andtrace element concentrations without anysample rangeofanalyticalmethods arenecessary(i) to assess the overall quality of preparation.Inorder to make immovable objects (e.g.archeologicalartifacts the products by comparison between batches, (ii)toidentifyaproductif in museums)accessible to LA-ICP-MS, it is desirable to uncouple the sam- counterfeit is suspected,or(iii) to evaluate the properties of apotential bio- pling processfromthe laboratorybased ICP-MS analysis.This workaimsat similar. establishing an analytical method for the determinationoftrace element Capillaryelectrophoresis (CE) is aseparationtechnique that is well suited to concentrations in ancient copperartifactswhichare not accessible via con- the analysis of therapeuticproteins thanks to itshighefficiencyand low ventionalLA-ICP-MSsetup. sampleconsumption. Highseparationperformance is reachedifissuessuch The sampling step is performedonsiteusing aportable nanosecond laser as adsorptionofthese biomolecules to the capillary walls, leading to poor device (λ=532 nm) andfiber optics to ablate the solidsample. The generated resolution andrecovery,are addressed [2]. The coupling of CE with mass aerosol is collected on polycarbonate membrane filtersand subsequently spectrometry(MS) enablesthe identificationofdegraded products.For this analyzed in alaboratorybased ns-LA-ICP-MS (λ=213 nm) setup. The useof purpose, MS-compatibleconditions have to be implemented. an externalstandard allows for trace element quantification. The objective of this workwas to develop an analyticalmethod basedonCE This analytical approach wasusedtoquantify trace element concentrations coupled to MS detection for filgrastim, abiotherapeutical drug used in can- in various copperreference materials. Good performances (±30%)were cer therapy. Experimental conditions were optimizedtoobtain minimized obtainedfor almost alltrace elements. Althoughmore accurate resultsand adsorption, separation of degradationproducts from the original protein, lowerlimitsofdetection can be reachedwhenanonline-LA-ICP-MSsetup compatibility with MS detection, togetherwith acceptable repeatability. is used, the attempt at offlinelaserablation sampling andsubsequent LA- ICP-MS analysis still remainsverypromising fortrace element quantifica- tion in samples that arenot accessible to aconventional LA-ICP-MSsetup. [1]Manning M.C.etal. PharmaceuticalResearch 2010, 27 (4),544. Following theapplication of the offline-LA LA-ICP-MSapproach to quanti- [2]HaselbergR.etal. Electrophoresis 2011, 32,66. fy traceelement concentrations in copper referencematerials,thismethod wasapplied to archeologicalcopperartifacts. 472 Chimia 2013, 67, Nr. 7/8 analytical sciences

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NMR Analysisofthe Components of the Reaction Mixture during the Isolation of naturalproducts:fromanalyticalHPLCtoMassSpec- Formation of the Glucose Oxime through Spectral Aliasing. trometry guided purificationbysemi-preparativeHPLC-MS

Karla Ramirez Gualito, Damien Jeannerat, AntonioAzzollini1 ,Emerson FerreiraQueiroz1 ,DavyGuillarme1 ,Jean- Organic Chemistry Department. University of Geneva. LucWolfender1 30, Quai Ernest Ansermet. 1211 Geneva, Switzerland. 1 School of Pharmaceutical Sciences,EPGL, UniversityofGeneva, Univer- Carbohydrates often exist in different conformations making them difficult sity of Lausanne,QuaiAnsermet30, 1211 Geneva,Switzerland to characterize.The chemical reactions of carbohydrates give rise to com- In naturalproductresearch theisolation of compoundsfromcrude extract is plex mixtures of derivatives with relevance such as oximes and hydrazones akey element. In this respect,the increase of purificationprocesseseffi- ciency,byimprovement of instrumental andmethodologicalapproaches,is which have found widespread applications, e.g. biomolecules labelling, ana- acrucial point. To tackle this issue, atwo-steps chromatographicstrategy lyzing protein−protein interactions and in vivo cell imaging. wasdeveloped.First,the separation of target moleculesfromcrude extract wasoptimized by linear gradient at theanalyticalscale.Second,the gradient Spectral aliasing is an NMR technique that improves the resolution in the wasgeometrically transferredtosemi-preparativeHPLCbyagradient trans- indirect dimension (F1) and can be accessed with areduction of the spectral fermethodbased on thecalibration of thechromatographicsystems (meas- urementofdwell volume andextra column volume)[1].UVaswellasMS window to the desired value monitoring were performedfor acomprehensive detectionofthe various compoundspresent within themixture.MSdetection proved to be an im- portanttool, notonlyfor thepurificationofcompounds that cannotbede- tected by UV or ELSD,but also foranefficient mass spectrometry guided purificationofspecificcompounds in crude extracts.Inparticular, asingle quadrupole mass spectrometercoupled with asemi-preparativechromato- graphicsystem(PuriFlash® -MS) wasfound apromisingtooltoincrease theefficiencyofconstituentsofinterestisolation.Thismassguidedsepara- tion of compoundsincomplex mixturerepresent apowerfulstrategy, not only forthe isolationofmoleculespresent in bioactivefractions,but also for therapid purificationofbiomarkersidentifiedbyUltraHPLC-MS metabo- lomics anddereplicationprocess. The aliased, 13C-HSQC recorded with 10 ppm in the 13Cdimension allowed acomplete assignment of 1Hand 13Cofall the components of the reaction [1]DavyGuillarme,Dao T.T. Nguyen,Serge Rudaz, Jean-Luc Veuthey, Eur. J. Pharma. and Biopharma. 2008 ,68,430 mixture without any further purification.

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Zooming in on the glucose metabolism of S. Cerevisiae. OMA andOPA -SoftwareTool forMass Spectra Interpretation of Natural andModified Nucleic Acids Alfredo J. Ibáñez1,Mareike Schmidt,2 Reinhard Dechant,3 Matthias Heine- mann,4 and Renato Zenobi1 Silvan R. Stucki,Adrien Nyakas,LorenzC.Blum,Jean-Louis Reymonds andStefan Schürch 1ETH Zurich, Department of Chemistry and Applied Biosciences, CH-8093 Zurich, Switzerland. 2Institute of Molecular Systems Biology,ETH Zurich, DepartmentofChemistryand Biochemistry, UniversityofBern, Bern, CH-8093 Zurich, Switzerland. 3Institute for Biochemistry,ETH Zurich, CH-8093 Switzerland Zurich, Switzerland. 4University of Groningen, Groningen Biomolecular Sci- ences and Biotechnology Institute, 9747 AG Groningen, The Netherlands Synthetic oligonucleotidescomprising unnaturalbuilding blockshave gained attention for human therapeuticapplications. Due to the presence of modifiednucleotides, the oligonucleotides exhibitincreased biostabilityand Even in normalized cell culture, cell populations show substantial cell-to- bioavailability. On the other hand, cellular DNAand RNAserve as targets cell differences in anumber of parameters including gene expression and for various chemotherapeuticdrugs, whichinterfere with transcriptionaland physiological parameters such as stress resistance and growth rate. This cel- translationalprocesses (e.g.antisense, RNAi). Tandem mass spectrometryis lular behavior can drastically influence the response of acell population to perturbations such as calorie restriction. the method of choice for sequencingofunnaturaloligonucleotides andelu- cidation of structuralalterations. Such experiments generate complex data Important processes associated with cell growth and calorie restriction in- sets, whose interpretation arelaborious andrequiresprofound knowledgeof volve transcriptional regulation. We wish to extendthis knowledge by using the gas-phase dissociation of nucleic acids. our single-cell metabolomic study platform called Microarrays for mass The software tool OMA &OPA hasbeen developed for interpretation of spectrometry (MAMS) to identify metabolic changes at the single cell level. production spectraofnatural andmodified oligonucleotides andtheirad- ductswithmetal ions andorganometallic drugs. Here, we present the our preliminary results from the comparison between metabolitesfrom individual yeast cells treated with (i) glucose-restricted, and (ii) normal cell culture medium; as well as cells grown in normal cul- ture medium that present different growth rates. We also present here how selected MS signals from the central metabolism can be used to better un- derstand stress resistance and differences in growth rate.

Nyakas et al., J. Am. Soc Mass Spectrom. 2012:p.1-8. analytical sciences Chimia 2013, 67, Nr. 7/8 473

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Microarrays formass spectrometry:Selfaliquoting nanoliter reactor Multiplexing magingLaserblationMassytometry plates fornano-LC-MALDI-MS applications at Sub-cellularSpatial esolution

* * a,c b c a Martin Pabst1 ,SimonKüster1 ,StephanFagerer1,Konstantins H.A.O. Wang, C. Giesen, D. Grolimund, B. Hattendorf, b a Jefimovs2,Petra Dittrich1,RenatoZenobi1 B. Bodenmiller, D. Gnther

1Department of Chemistryand AppliedBiosciences,ETH Zürich,Wolfgang a Trace Elementand MicroAnalysisGroup, ETHZurich, 8093 Zurich PauliStrasse 10,8093 Zürich,Switzerland b Institute of MolecularLifeSciences, UniversityofZrich, 8057 Zrich 2LaboratoryofElectronics/Metrology/Reliability, EMPA,Swiss Federal c microXAS Beamline Project,Swiss LightSource,PSI,5232 Villigen PSI Laboratoriesfor Material Sciences andTechnology,CH-8600 Dübendorf, Equalcontribution Switzerland We describe in thisworkanadvanced multiplexing imagingsetup In this work, self aliqoutingmicroarrayplates[1] are usedasasample target basedonLaser Ablation(LA)coupled to acommercial mass cytometer fornanoliterfractions collected by ahigh-frequencydroplet spotter [2], (CyTOF).The currentmeasurements successfully demonstratesub-cellular whichisconnected to ananoliquidchromatography system. (1m) spatialresolutionimaging of biological tissue thin sections. Oneof Fractions of thenano-LC (with flow rates of 250-500 nl/min)are collected thekey componentsofthe setupisanovelLAcell, providing shortwashout in 1Hzrates anddepositedintothe micro-cavities. The hydrophobiccoating time andenhancingthe signal to noise ratio of theLAtransientsignal.1 This of theplatesconfines thedropletstothe predefinedposition. Thecavities enablescompleteseparationofion signalsfromindividualpulsesgenerated furtherallownanolitervolumereactions directly on themicroarrayplate by 20 Hz laserablation andfurthermoreacquisitionofanalytesinthe sample itself.Upto2700 spotscan be collected on aplate in thedimension of a aerosol produced by 1mlaser single shot. As showninacase study, mul- standard microscopeslide (thiscorresponds to 45 minutes collectingtime,if tiple biomarkers,metal-taggedantibodies were imagedsimultaneouslyin working at 1Hzrates). thin sections of breastcancertissue,using this experimental setup. The re- Peaksfromanano LC reversed phasecolumn(3µm, 75µmx10cm) are usu- sultinghighresolutionbiomarker imageshelpbiologiststoinvestigate ally spreadoveratleast10spots, andthe repetitiveinformationcan be fur- various breastcancersub-types,and analyzecell-cell interactions.2 The pre- ther usedfor an integrated enzymatic or chemical digest of thetargetana- sentedimaging setupwillopennew research opportunitiesfor pathologists lytes, either before or aftermassspectrometric analysis. Thiswas demon- andpharmacologists andsomeofthe potentialapplications will be discussed. strated by collectingthe eluate fromareversed phaseseparationofatryptic proteindigest, in ordertodevelop areliableway foridentificationofpost 1Wang, H.A.O.,etal. Fast Chemical ImagingatHighSpatial translational modifications. Theself-aliquotingpropertiesofthe microarray Resolution by LaserAblationInductivelyCoupled Plasma Mass plates allows furtheramanualpre-aliquotingofmatrix, internal standardsor Spectrometry. SumittedtoAnaltical Chemistr (2013).

even enzyme solutions -byjust using asimple mechanical sliding device. 2 2Giesen,C., Wang,H.A.O., Hattendorf, B.,Grolimund, D.,Gnther, [1]P.L.Urban et.al.,Lab.Chip.,2010 D.,BodenmillerB.etal. Sumitted (2013). [2]S.K.Küster et.al.,Anal. Chem., 2013 *both authorscontributedequally to this work

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Monitoringoffluorescentlabeling reactions IncrementalModel Identification of Gas-LiquidReactionSystems with Unsteady-State Diffusion Julien Héritier, YvanCrittinand Jean-Manuel Segura Benoit Cretegny, Sriniketh Srinivasan, Julien Billeter, Dominique Bonvin InstituteofLifeTechnologies, HES-SO Valais, RteduRawyl 64, 1950 Sion Ecole Polytechnique Fédérale de Lausanne (EPFL) Fluorescence detectionormicroscopyofbiomolecules oftenrequiresprior Laboratoire d’Automatique, Switzerland labeling with efficient fluorophores.For proteindetection in particular, isothiocyanate or succinimidylester derivativesoffluorophores such as flu- Identification of kinetic models and estimation of reaction andmass-transfer oresceinare widely used to covalentlyreact with the -aminogroup of ly- parameters can be performedusing theextent-based identification method, sines on proteins. Despitetheir broaduse in bioanalytics,few reports have whereby each chemical/physical process is handled separately [1-3]. This investigated thekinetics of these reactions undervarying conditions, alt- method is used here to analyze gas-liquid systemsunder unsteady-state houghthiswould allow optimization of labeling protocols. mass transfer. Such asituation is common in the case of diffusion-controlled reactions and is modeled by the film theory, that is, transferring speciesac- Here we show that fluorescentlabelingreactions can be monitoredusing cumulateinaliquid film. In both thegas and liquidbulks, mass-balance fluorescence-polarizationdetection. The attachment of the small fluorophore relationsdescribe the speciesdynamics as ordinary differential equations to alarge protein resultsinanincreaseinfluorescence anisotropy, which (ODE) and serve as boundary conditions for the film.Onthe other hand, the can be followedovertime. The kinetics of severallabelingreactions was dynamic accumulation in the film is described by Fick’s second law. The determined as afunction of pH, concentrationand moleratio of reagents. resulting partialdifferential equation (PDE) systemissolved by discretiza- Furthermore,the impact of side reactions such as hydrolysis andfluoro- tionand rearrangementinODEs. phore dimerizationwas assessed. The estimationofdiffusion coefficients follows atwo-steps procedure. First, the extents of mass transfer arecomputed from measurements in thetwo bulks. Diffusioncoefficients are then estimated individually by fittingeach extent of mass transfer to the extent obtained by solvingthe corresponding PDE. Comparison of the estimated diffusioncoefficients with their literature values serves to validate themodels identified in the two bulks.

The estimation of both kinetic parameters and diffusion coefficients is in- vestigated forgas-liquid reactionsystemswith unsteady-state diffusion. The approach is illustratedwith simulated examples.

[1]Bhatt et al,Ind.&Eng.Chem. Res. 50, 12960-12974, 2011 [2] Srinivasan et al, Chem. Eng. J. 208, 785-793, 2012 [3] Billeteretal, Anal. Chim.Acta 767, 21-34, 2013 474 Chimia 2013, 67, Nr. 7/8 analytical sciences

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Incremental Model Identification using the Concept of Extents Evaluation of superchargingmolecules with thesheathliquidinterface in CE-ESI-MS Sriniketh Srinivasan, Julien Billeter, Dominique Bonvin

Ecole PolytechniqueFédérale de Lausanne (EPFL) Grégoire Bonvin,Florent Clerc, SergeRudaz, JulieSchappler Laboratoire d’Automatique, Switzerland School of Pharmaceutical Sciences,UniversityofGeneva, Universityof Kinetic models contribute greatly to cost reduction during the process de- Lausanne,Bdd’Yvoy20, 1211 Geneva 4, Switzerland velopment phase and are also helpful for process monitoring and control purposes.Kinetic models describe the underlying reactions, mass transport and operating conditions of the reactor. In the typical one-step simultaneous Capillaryelectrophoresis (CE) coupled with electrospray ionization (ESI) method of identification, one postulates adynamic model encompassing the mass spectrometry(MS) is asuitable technique for analysis of intact pro- effects of all phenomena at stake, and model parameters are estimated by teins.The main configurationtorealize thiscoupling is the sheath liquid comparing measured data with model predictions. Simultaneous identifica- interface whichisconstituted of two concentric stainlesssteeltubes. The tion can be computationally costly and exhibit convergence issues in case of first one surrounds the free endofthe CE capillarywhere asheath liquid poor initial guesses. Furthermore, this method is characterizedbyhigh cor- provides electric contact,appropriateflow andsolvent composition for ioni- relation between parameters, which can lead to structural mismatch. zation andevaporation. The second one permits to introduceanebulizing gastoassist the sprayformation.Generally,the ionization of proteins and In contrast, the extent-based incremental method of identification is atwo- especially theirchargestate distribution (CSD) is mainlyinfluenced by their step approach, in which measured data are first transformed into extents, conformation as well as the nature andcomposition of the ESI effluent. each one representing the effect of aparticular phenomenon [1-3]. Then, for Thus, the sheath liquid hasadirect impact on ionization andamodification each phenomenon individually, amodel is postulated and the corresponding of itscomposition can change the protein CSD.Because this interface is parameters estimated by comparing the simulated and measured extents. highlyversatile, it can be used for modifyingionizationwithoutaffecting Since each extent, and thus each effect, is handled individually, the correla- CE selectivityaswell as apost-capillaryinfusion device. tion between model parameters is considerably reduced. In thiswork, the effect of the addition of supercharging molecules[1] to the sheath liquid on the CSD of proteins wasevaluated.Several supercharging This presentation will give an overview of the extent-based incremental agents such as nitro benzoylalcohols (NBAs) were testedonmodel proteins identification and will describe the procedure to analyze homogeneous and (i.e.insulin, growth hormone,hemoglobin) presenting differentpropertiesin gas-liquid systems. The performance of simultaneous and incremental termofionization,conformation andflexibility.The influenceofthese su- methods of identification will be compared via simulated examples. perchargingadditives on sensitivity(i.e.protein ionization efficiency) was also estimated. [1] Bhatt et al, Ind. &Eng. Chem. Res. 50, 12960-12974, 2011 [2] Srinivasan et al, Chem. Eng. J. 208, 785-793, 2012 [1]S.S.Miladinovic, L. Fornelli,Y.Lu, K. M. Piech, H. H. Girault,Y.O. [3] Billeter et al, Anal. Chim. Acta 767, 21-34, 2013 Tsybin, Anal Chem. 2012, 84,4647.

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Sol-based optical Ammonia Gas Sensor Study of thebehavior of individualmulti-element droplets in an induc- SusanneWidmer1,2,Marko Dorrestijn1,Edwin C. Constable2, tively coupled plasmabyhightemporalresolutionmass spectrometry LukasJ.Scherer1* O. Borovinskaya1,B.Hattendorf1,M.Tanner2,D.Günther1 1Empa, Swiss Federal Laboratories forMaterial Science and Technology, Laboratory forProtectionand Physiology, Lerchenfeldstr. 5, 9014 St. Gallen, Switzerland 1) Department of InorganicChemistry, ETH Zurich,Wolfgang-Pauli- 2University of Basel, Department of Chemistry, Strasse 10,Zurich, Switzerland Spitalstr. 51, 4056 Basel, Switzerland 2) Tofwerk AG, Uttigenstrasse 22,Thun, Switzerland

Stateofthe art gas sensors usuallyare hand-held devices. Thedevelopment Despitethe widespread acceptanceand use of inductivelycoupled plasma of agas sensor system based on flexible polymer optical fibers, which mass spectrometry(ICPMS) allthe processesthatananalyte undergoesin finally canbeintegratedinto textiles, would increase sensorwearability.As theICP are still poorly understood. Abetterunderstanding of these amodel system,sols made out of organicmodified silicatesdoped with processeswouldhelptoimprove instrumental performance andanalysis different mixtures of fluorescent coumarin andfluorescein were solvent accuracy. Combinationofasingledroplet introductionsystem[1] with a casted and dried on poly(methyl methacrylate) (PMMA)plates, which were prototypehightemporal resolutionICP time-of-flight MS allows exposed to gaseous ammonia. The combinationofthe two dyes showed a investigatinganalyte-plasmainteractions on thebasis of asingledroplet muchhigher sensitivity towardsammonia than eithersingledye. This signal.Atemporal separationofthe ionsignaloccurrences of different indicates thatthe Förster energy transfer betweenthese dyes is essentialfor elements fromasingle droplet,which hadbeen observed in theprevious thesensing mechanism. work [2], wasfurther investigated in this study. Mixtures of differentsalts Significant differences in fluorescence intensity changesat430 nm were andparticleswere analysed.Itwas shown that theorder of occurrence of the observed for different dye mixtures, xerogel thicknesses and gas elements’signalmaxima correlates with themelting/boilingtemperatures of concentrations(Figure). thecorresponding oxides [3]and can be changedbythe central gasvelocity. 5 The obtained results indicatethatthissignalseparationisdue to thespatial 4.5 separationofelements, taking placeduringthe transitofadroplet in the

4 100 ppm NH3 ICP. Moreover, it wasdemonstrated that particle composition, plasma V]

e[ 3.5 parameters andthe axial/radiallocationofanalyte vaporizationonsethavea 1000 ppmNH

ag 3

lt 3 directinfluence on thespatialseparationofelements. Apossible explanation

Vo 2.5 forthiseffect andits implications on thefinal intensity andstabilityofthe 2 signal will be discussed. 05000 10000 15000 20000 Time [s] [1]Gschwind et.al.,JAAS, 2011, 26, 6,1166. Figure: Dye doped 27 µmthick xerogel periodically exposed to 100 or [2]Borovinskaya et.al.,JAAS, 2013, 28, 2,226-233. 1000 ppmNH(g) (loweringinvoltage)and N (g)(increaseinvoltage). 3 2 [3]Flamigni et.al.,JAAS, 2012, 27, 4,619-625. analytical sciences Chimia 2013, 67, Nr. 7/8 475

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Advanced Analysis of Post-Translational Protein Modifications Using a Signal processing and instrumentation for Fourier transform nano-LC-MALDI-MS InterfaceBased on Droplet Microfluidics mass spectrometry with improved analytical performance

Simon K. Küster*, Martin Pabst*, Renato Zenobi and Petra S. Dittrich Anton N. Kozhinov,1 Tagir Aushev,2 Yury O. Tsybin1

Department of Chemistry and Applied Biosciences, ETH Zurich, 1Ecole Polytechnique Fédérale, 1015 Lausanne, Switzerland 8093 Zurich, Switzerland 2Institute of Theoretical and Experimental Physics,117218Moscow, Russia

Post-translational modifications (PTMs) are highly relevant for regulat- Fourier transformmassspectrometry (FTMS)-based applications necessitate ing the properties and functions of proteins. However, with conventional improved analytical performance. Thecurrent limitations in resolution, analytical methods, it is difficult to locate and identify these modifications. mass accuracy, measurement precision, and other analytical characteristics To address this challenge, we have developed anovel droplet-based micro- of FTMS are imposed by the conventionalsignal processing and instrumen- fluidic interface that combines separation by nano-liquid chromatography tation. Herein,weevaluatethe filterdiagonalization method (FDM),which (LC) with detection by matrix-assisted laser desorption/ionization mass providesincreased resolution compared to that of the conventionalsignal spectrometry (MALDI-MS). The system facilitates not only enhanced ali- processing. Specifically, the resolution, mass accuracy, and dynamic range quotation of eluted peaks at yet unmatched temporal resolution but also in- of FDM-based FTMSare characterized. Further,weimplement the least- troduces an additional enzymaticaldimension integrated into the standard squares fitting(LSF) method,whichreveals the instrumentation-limited LC-MS analysis workflow.This allows reliable detection of protein modifi- levels of achievable analyticalcharacteristics. Specifically,the LSF allows cations and also the identification of the nature of the detected modification. studying the effect of length and formofthe transient signal, thermal noise Droplet microfluidics is used to compartmentalize the eluate from the of thedetector, sampling rate of the digitizer, synchronization precision of nano-LCinto astream of nanoliter droplets at high frequency. The droplets theinstrumentelectronics, electrodes geometryofthe mass analyzer, and are spotted on amicroarray with 2780 hydrophilic spots of 300 µm diameter spatial spread of the ion package. Optimization of these parameters is con- on ahydrophobized microscope slide as described previously [1]. Due to the sidered in ordertoincrease theachievable levels of FTMSanalytical charac- high sampling frequency of our spotter, typically 1Hz, each eluted peak is teristics. Finally, we implement the high-performance data acquisition sys- aliquoted into 10 or more spots. Hence, each of these 10 or more spots con- tem, which allows high-definition acquisitionofexperimental transient sig- tains the same analyte. By depositing asecond nanoliter droplet to every nals from ions in themass analyzer.Experiments were performed on 10 T other spot we then perform nanoscale reactions. The added droplet contains LTQ FT-ICR MS and LTQ OrbitrapEliteFTMS(ThermoScientific). The an enzyme which selectively removes the PTMs from the analyte. Using obtained results advancethe MS-based high-resolution molecular structure MALDI-MS, we are then able to reliably detect peptide modifications such analysis of complexmixtures via increased resolution, mass measurement as glycosylation or phosphorylation just by performing apairwise compari- accuracy, andsignal-to-noise ratio.Particularly, we considerapplicationsof son of mass spectra obtained from neighboring spots. developedmethods and techniques in peptideand proteinstructure analysis, structural biology, and analysis of petroleum-type samples. [1] S.K. Küster, S.R. Fagerer, P.E. Verboket, K. Eyer, K. Jefimovs, R. Zenobi, P.S. Dittrich, Anal. Chem. 2013, 85,1285