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Analytical Sciences 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 mass spectrometry 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, chemistry, 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. Analytical Sciences As03 Analytical Sciences As04 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 Ions 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 5 te 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 Analytical Sciences As05 Analytical Sciences As06 Microwave Plasma –AtomicEmission Spectroscopy: 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 chromatography 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. Analytical Chemistry As07 Analytical Sciences As08 Advances in azide-alkynecycloaddition (click-chemistry) based manu- Fluorescence
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