RNA Epitranscriptomics and Proteomics Resource

Analysis by Agilent LC-MS QTOF

Standard operating procedure (SOP)

Author:Dr.VladimirErmolenkov

Date:04/23/2018

Summary:ThisSOPdescribestheapprovedproceduresforperformingself-serviceanalysisontheAgilentLC-MSQTOFinstrument.Failuretofollowtheseproceduresmayresultnotonlyinunsuccessfulanalyses,butalsoinperformancedeteriorationandinstrumentdamage.FailuretocomplywithSOPandsafetyruleswillresultinthelossofself-serviceprivileges.SamplepreparationproceduresaredescribedinseparateadhocSOPs.

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I. Purpose:ThepurposeofthisdocumentistodescribetheacceptedproceduresforusingtheAgilentLC-MSQTOF.

II. ScopeandImplementation:ThisSOPappliestoAgilentLC-MSQTOFinstrumentlocatedintheLifeSciencesResearchBuilding,roomLS1151.

III. Introduction:TheAgilent6530BAccurateMassQuadrupleTime-of-FlightmassspectrometerisequippedwithdualelectrosprayionizationsourceandanAgilent1260UPLCliquidchromatograph.Thesystemwasdesignedforprofiling,identifying,andcharacterizinglowmolecular-weightcompoundsandbiomolecules.Itcanprovideamassresolutionofupto20,000andstandardmassrangeofupto3,200m/z.Theinstrumentcanbesetuptoaffordanextendedmassrangeofupto20,000m/z.TheAgilent’sMassHunterWorkstationsoftwarefacilitatesprocessingofdatageneratedbytheAgilentLC-MSQTOF.

IV. Definitions:

a. MassSpectrometer(MS)System:ForthepurposesofthisSOP,anMSsystemisdefinedasallcomponentsrequiredforgeneratingusablemassspectral-baseddataforagivensample.ThisincludesbutisnotlimitedtoAgilent1260liquidchromatograph,allelectronics(powersupplies,amplifiers,digitaltoanalogconverters,etc.),allhardware(lenses,massanalyzers,vacuumpumps,etc.),andallassociateddatasystemhardwareandsoftware.

b. REPRStaff:amemberoftheresourcewhoisaninstrumentspecialistinchargeofregularoperations,troubleshooting,andmaintenanceoftheMSsystem.Thispersonisresponsiblealsoforprovidingspecifictrainingandconsultationsoninstrumentusage/operations.

c. User:Personwhoreceivedspecifictrainingontheusage/operationsoftheMSsystem,obtainedpermissiontoperformself-serviceanalysis,andreceivedanaccountforbookinginstrumenttime,record-keeping,andbilling.ThispersonisresponsibleforfollowingtheSOPtoacquirethedesiredMSdataforagivensampleorsetofsamples.

d. Analyticalmethod:Steps(s)tobefollowedforagivenanalysisofagivencompound(s)inagivenmatrix,includinganoutlineofspecificoperationalparametersforanMSsystemusedinperformingsaidanalysis.

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V. Responsibilities:a. ItistheUser’sresponsibilitytobefamiliarwithallstandardlaboratorysafety

procedures.Additionally,itistheUser’sresponsibilitytobefamiliarwithinstrument-specifictrainingonsafeoperations,whichisprovidedon-demandbyREPRStaff(videinfra).NopersonisallowedtoapproachtheMSsystemwithoutreceivingspecifictraining/certificationfromthedesignatedREPRStaff.

b. ItistheUser’sresponsibilitytocoordinatewithdesignatedREPRStaffanynecessaryhardwareswitchoverandsetup,anytroubleshooting,repair,maintenance,calibration,anddocumentation.UnlessexplicitlyauthorizedbyREPRStaff,theUserisnotallowedtochangethecurrenthardwareconfiguration.

c. ItistheUser’sresponsibilitytoreserveinstrumenttime;tomakeajudicioususeofinstrumentavailability;andtorecordrequiredinformationabouttheanalysisintheSampleLogbookandaboutthestatusoftheinstrumentintheAgilentLC-MSQ-TOFlogsheets(videinfrafordetails).

d. UsersareresponsibleforrinsingtheLCpartofthesystemwiththedesiredmobilephase;runningtherecommendedstandardbeforetheactualanalysis;adheringtotheindicatedlimitsofsampleconcentration;rinsingtheLCpartofthesystemwiththeindicatedphase,afterrunningsamples.

e. UsersmusttakeandsavescreenshotsoftheMassHunterDataAcquisitionsoftware,includingthechromatogramplotwiththetotalioncurrent(TIC)curve,thespectrumplotwiththecurrentmassspectra,theActualsplotwiththeBinaryPumppressure,andthedate/timeoftakingthescreenshotbeforeandafterexperiments.Thefirstscreenshotshouldbetakenandsavedbeforeinjectionofthefirstsample.If

1)theTICinChromatogramPlotafterinjectingthelastsampleandwashingtheLCishigherthanbeforethefirstinjection,or

2)thecurrentmassspectrainSpectrumPlotcontainextrabandsthatwerenotpresentbeforethefirstinjection,or

3)theBinaryPumppressureishigherthanbeforethefirstinjection,

theUsermustcontinuewashingtheLCuntilTICandtheBinaryPumppressuredroptothebefore-injectionlevelandtheextrabandsinthemassspectradisappear.WhentheappropriateTICandthemassspectraarereached,theUsermusttakeandsaveanotherscreenshotasaproofofcleanlinessoftheLC.

VI. Training:

Allpersonnelmustbetrained/certifiedbyqualifiedREPRStaffbeforeusingtheAgilentLC-MSQTOF.Additional information, tips,or requirementsmaybeprovidedata latertimetoensurecontinuedproperuseof themassspectrometer.Usersarerequiredtobecome familiar with this SOP and pertinent sections of instrument’s manuals) videinfra).

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VII. SchedulingandBilling:

Instrument time must be reserved in advance by using the Faces web-server athttps://faces.ccrc.uga.edu/. An individual account will be created for eachtrained/certified User. To encourage fairness of signups and frugality of usage, eachUserwillbebilledaccordingtothetimereservedonFaces.TherationaleforthismodusoperandiisexplainedintheREPRServicePoliciesdocumentavailableontheresource’swebsite.

VIII. Samplelogging:Users must record pertinent information on the instrument’s Sample Logbook. TheinformationincludesnotonlytheUser’snameandlab,butalsotheputativenatureandconcentrationofthesample.EachUserisrequiredtoincludealsothem/zofthemostintensesignalinthesampleorsampleset,whichwillallowREPRStafftotrackpossiblecontamination.

IX. Documentation:

A service log is maintained for each REPR’s MS system, which contains all servicereports.Recordsofmasscalibrations,analysisofapprovedstandards,andanydataonsystem performance are kept on the hard drive of the computer controlling theMSsystem(videinfra).

X. Instrumentmanuals:Electroniccopiesofthemanufacturer’smanuals,whichincludedetaileddescriptionsofthe operations of the MS system, are located on the hard drive of the computercontrollingtheMSsystem.

XI. Procedures:a. Whenfirstapproachingthesystem,theUsershouldcheckvariouscomponentsof

theMSsystemtoensurethatitisreadyforregularoperations.Thechecklistincludes:

1- Vacuumsystem:checkthepressurereadingsintheActualspaneintheMassHunterDataAcquisitionsoftware.AlertimmediatelytheREPRStaffifthepressurereadingsarenoticeablydifferentfromthoseshownbelow.

2- Masscalibration:calibrationandresolutionareregularlycheckedbyREPRStaff,

whoperforminstrumentcalibrationonanas-neededbasis.Thisoperationis

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carriedoutbyusingtheAgilentTuningMix(productnumberG1969-85000),whichgeneratesionsofknownm/zratioslistedintheMassHunterDataAcquisitionsoftware.Beforeinitiatingtheanalysis,itistheUser’sresponsibilitytoinquirewiththeREPRStaffaboutthecurrentstatusoftheinstrument’scalibrationandresolution.

3- UPLCliquidpressure:checkifthebinarypumppressureiswithinnormalrangefortheAgilent1260UPLC.

4- Chromatographicinletsshouldbecheckedforrequiredquantityandtypeofcarrierfluidandanyleaks.Wheneverproblemsarefound,alertREPRStafftorectifythempriortosampleanalysis.

5- REPRStaffisresponsibleforinspectingandcleaninginletinjectorassemblies,ionsourceassemblies,iontransfercapillary,lensassemblies,etc.UsermustreporttoREPRStaffanydeteriorationofnormalsystemperformance,asindicatedbypoormasspeakshape/resolutionand/orpresenceofcontaminantbandsinmassspectra.

b. AnnualProcedures:REPRStaffisresponsibleforscheduling/performingpreventivemaintenanceat12-monthintervals.Preventivemaintenanceitemsaredeterminedbasedonthemanufacturer'srecommendationfortheMSsystem.ItistheUser’sresponsibilitytoinquirewiththeREPRStaffaboutpossibledisruptionofinstrumentavailabilityduetoannualpreventivemaintenance.

XII. SamplePreparation:AseparateSOPisavailableforeachtypeofself-serviceanalysisallowedontheAgilentLC-MSQTOFinstrument.Thefollowinggeneralprinciplesapply:- Usersareexpectedtopreparesamplesintheirownlaboratoryandtransferthemto

theinstrumentroomreadyforanalysis.

- Samplesconcentrationsarenotsupposedtoexceed0.5mg/mLformoleculesbelow1,000Da,or0.1mg/mL for largerones.Greaterconcentrationsmaycausesystemcarryoversandsourcecontamination,withundesirablelossofperformance.Samplesolutions are not supposed to contain buffers based on alkaline or alkaline-earthcations

- Failuretocomplywiththisrequirementwillresultinlossofself-serviceprivileges.

- Unlessotherwiseagreedupon,REPRwillprovideproperHPLC-gradesolvents.

LC-MSgradeacetonitrile andmethanol andnanopureDIwaterwith0.1% formic acidand5%acentonitrileaddedareprovidedasmobilephasesforUPLC.UserscanusetheirownvolatileLC-MSgradesolventsintheirownbottles.Non-volatilesolventslikeDMSOandDMFarestrictlyprohibited forusingasmobilephases!Theuser’s solventbottles

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mustbecleanedwithNitricacidandrinsedwithat least fivevolumesofnanopureDIwaterpriortouse.Samples• Samplesshouldbepreparedintheuser’slab.• HPLCorhighergradesolventsmustbeusedforthesamplepreparation.• Saltconcentrationinthesamplesshouldbeminimizedreasonably.• The recommendedconcentrationof samples is<andof largemolecule samples is

<0.1mg/ml.• All samples must be filtered through a 0.45 micron or finer filter just prior to

analysis.Noexceptions!Ignoringthisrulewillcausesuspensionofprivileges.

XIII. LCColumnsUsersmust provide their own LC columns, as appropriate for their project. REPR canprovidetheonlyC18columnforseparationofsmallmoleculesandshortpeptideswithMW<5kDa.

XIV. StepwiseSampleAnalysis:DataAcquisition1.SignintheAgilentMassSpectrometerLogSheet.2. Locate themobilephasebottles in thebottle trayon the topof theLC.Makesurethat the solvent filters are located in the bottles with the right solvents for yourexperiment. Otherwise, swap the bottles. Remember, that the filter with the tubingconnectedtotheLCchannelAmustbeinthebottlewithwaterandthefilterwiththetubing connected to the LC channel B must be in the bottle with organic solvent(acetonitrileormethanol).Alwaysweargloveswhenyouswapthebottles!

3.Placeyoursamplesintheavailableslotsoftheautosamplerplate.

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4.Loginthecomputer5.ClickontheDataAcquisitionicontoopentheMassHunterDataAcquisitionsoftware.

6.OpenyourmethodinMethodEditor,modifyitforyourexperiment,andclickontheApplybuttonnext to themethodname field. If youdonothaveamethod,open theTemplate.mmethod,modifyitforyourexperiment,clickontheApplybutton,andsaveunder some other name. The template method is located in theD:/MassHunter/Methods folder. Always click on the Apply button when you makechangesinthemethod!

7.Set thespectral rangeto90-1000m/z inMethodEditor/QTOF/AcquisitionandclickApply.

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8. Turn on the instrument by clicking on the greenOn icon in the Instrument Statuspanel.

9.Waitfor5minutes.10.DirecttheLCstreamtoMSinMethodEditor/QTOF/General/LCSream(Seg).Todothat,clickontheMSradiobuttonandclickApplyNow.

11.FindtheSnippingToolintheStartMenuandtakeasnapshotofthesoftwarescreenincluding Chromatogram Plot, Spectrum Plot, Actuals, and current date/time in thebottomrightcornerofthescreen.

12.SavethesnapshotasaJPGfileintheD:/Screenshots/Today’sDatefolder.Givethefileaname including today’sdate,current time,andyourname.For instance, for thefile saved on April 05, 2018 at 9:00 am by Vladimir, the file name could be“20180405_9_00am_Vladimir.jpg.”13. Set the spectral range inMethod Editor/QTOF/General (see the picture in p.7) inaccordancewiththeprotocolofyourexperiment.

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14.SetupSampleRunorWorklist.

15.ClickontheStartbuttoninSampleRunorWorklisttorunyoursample(s).

16. Repeat p. 13-15 for all samples. Run the samples with at least 5-minute intervalbetweentherunsforcleaningtheLC.17. When the last run is complete, direct the LC stream to Waste in MethodEditor/QTOF/General/LCSream(Seg).Todothat,clickontheWasteradiobuttonandclickApplyNow.Keepthe instrument inthisstate forat least10minutes forwashingtheLC.

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18.Setthespectralrangeto90-1000m/zinMethodEditor/QTOF/AcquisitionandclickontheApplybutton(seethepictureinp.7).19.DirecttheLCstreamtoMSinMethodEditor/QTOF/General/LCSream(Seg).Todothat,clickontheMSradiobuttonandclickApplyNow(seethepictureinp.10).20. Compare the TIC value in Chromatogram Plot, themass spectra in the SpectrumPlot,andtheBinaryPumppressureinActualswiththoseinthepicturesavedinp.12.IfTIC inChromatogramPlotandtheBinaryPumppressurearehigherthanthoseonthepictureand themass spectra in theSpectrumPlot containextrabandswhicharenotpresent on the picture, direct the LC stream to Waste in MethodEditor/QTOF/General/LCSream(Seg)(seethepictureinp.17)andcontinuewashingtheLC.

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21.Repeatp.19-20periodicallyuntilTICandtheBinaryPumppressurebecomeequaltothecorrespondingvaluesbeforeinjectionofthefirstsampleandtheextrabandsinthemassspectrainSpectrumPlotdisappear.22.DirecttheLCstreamtoMSinMethodEditor/QTOF/General/LCSream(Seg).Todothat,clickontheMSradiobuttonandclickApplyNow(seethepictureinp.10).22 Using the Snipping Tool, take the snapshot of the software screen includingChromatogramPlot,SpectrumPlot,Actuals,andcurrentdate/timeinthebottomrightcornerofthescreenandsaveitintheD:/Screenshotsfolder(seep.11-12).23.ClickontheredOffbuttontoputthemassspectrometerintheStandbyregime.

24.ClosetheMassHunterDataAcquisitionsoftware.

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When themessageswith the questions pop up, answer Yes to the question “Do youwishtoputtheinstrumentinstandby?”andYesorNotoallotherquestions.

25.SignoutintheAgilentMassSpectrometerLogSheet.For more detailed information on the data acquisition procedure, read “G3335-90174_TOF_QTOF_QuickStart”guide.DataProcessingData processing is accomplished using the MassHunter Qualitative Analysis (Qual)software.

To learn Qual, watch the training videos located in Desktop/Software TrainingVideos/VideoTutorialforMassHunterQualitativeAnalysisfolder.

XV. Safety:

Generalinstrumentsafety.PHYSICALINJURYHAZARD:UsetheLC/MSinstrumentonlyasspecifiedinthisSOPandtheinstrumentmanuals.Usingthesysteminanotspecifiedmannercanresultinpersonalinjuryordamagetotheinstrument.Solvents.PHYSICALINJURYHAZARD:Alwayswearappropriateprotection,includingeyeprotectionandgloves,whenworkingwithsolventsandchemicals.Refertothe

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applicableMSDSformoreinformationaboutthematerialsyouareworkingwith.ElectricalSafetyWhentheLC/MSinstrumentispluggedintoapowersource,evenifthepowerswitchisoff,dangerousvoltagescanexist:•InthewiringbetweentheLC/MSinstrumentpowercordandtheACpowersupply.•TheACpowersupply•InthewiringfromtheACpowersupplytothepowerswitch.If thepowercord insulation isdamaged, frayed,orworn, let the instrumentmanagerknowimmediately.Neverremovetheinstrumentcoverunlessspecificallyinstructedtodoso.Neverdoanyworkatelectricalparts.TemperaturesafetySomepartsoftheLC/MSinstrumentoperateattemperatureshighenoughtocauseseriousburns.AlwayscoolheatedpartsoftheLC/MSdowntoroomtemperaturebeforeyouworkonthem.Tocooltheinstrumentfaster,settheheatedzonetoroomtemperature.Turnthezoneoffafterithasreachedthesetpoint.Ifyoumustdoanyworkonahotpart,alwaysweargloves.ChemicalsafetyBeforehandlinganychemicals,refertotheMaterialSafetyDataSheet(MSDS)providedbythemanufacturer,andobserveallrelevantprecautions.Tominimizethehazardsofchemicals:

•ReadandunderstandtheMSDSprovidedbythemanufacturerbeforeyoustore,handle,orworkwithanychemicalsorhazardousmaterials.•Minimizecontactwithchemicals.Wearappropriatepersonalprotectiveequipmentwhenhandlingchemicals(forexample,safetyglasses,gloves,orprotectiveclothing).•Minimizetheinhalationofchemicals.Donotleavechemicalcontainersopen.Useonlywithadequateventilation(forexample,fumehood).•Checkregularlyforchemicalleaksorspills.Ifeitherofthoseoccur,followthecleanupproceduresfromthemanufacturer,asrecommendedintheMSDS.•Complywithalllocal,state/provincial,ornationallawsandregulationsrelatedtochemicalstorage,handling,anddisposal.

ChemicalwastesafetyCHEMICALWASTEHAZARD:WastesproducedbytheLC/MSSystemarepotentiallyhazardousandcancauseinjury,illness,ordeath.

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Tominimizethehazardsofchemicalwaste:•ReadandunderstandtheMSDSprovidedbythemanufacturersofthechemicalsinthewastecontainerbeforeyoustore,handle,ordisposeofchemicalwaste.•Usewastecontainerscompatiblewiththewastematerialandmeetingfederal,state/provincial,andlocalrequirementsforcontainerstorage.•Minimizecontactwithchemicals.Wearappropriatepersonalprotectiveequipmentwhenhandlingchemicals(forexample,safetyglasses,gloves,orprotectiveclothing).•Minimizetheinhalationofchemicals.Donotleavechemicalcontainersopen.•Handlechemicalwastesinafumehood.•Afteremptyingawastecontainer,sealitwiththecap.•Disposeofthecontentsofthewastetrayandwastebottleinaccordancewithgoodlaboratorypracticesandlocal,state/provincial,ornationalenvironmentalandhealthregulations.•EnsurethattheLC/MSinstrumentwasteisstored,transferred,transported,anddisposedofaccordingtoalllocal,state/provincial,and/ornationalregulations.