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PNNLPreProcessor|IonmobilityMSUSERGUIDE–version2017.08InclosecollaborationbetweenPacificNorthwestNationalLaboratoryandAgilentTechnologies,wehavedeveloped thiseasy-to-use software toolwhichdirectly readsAgilentMassHunter (.d)data filesandgeneratesnew.ddatafileswithenhancedsignalquality.
Availablepreprocessingmethodsincludemultidimensionalsmoothingandfilteringofrawdatafromionmobility-mass spectrometry (IM-MS) analyses. Functionality to repair saturated peakswill be addedsoon.
1. Whatarethebenefits?• Removeartifactsinjaggedpeaks(i.e.lowionstatistics)andenhancelow-abundancerealsignals.• Improvesignal-to-noiseandreproducibilitywithmorepeaksconsistentlydetectedacrossyour
multipleruns.• Reducevariationsinabundanceandcollisioncrosssectionvalues.• Reducethefilesizeandtimefordownstreamprocessing.• Computationallyextendthedynamicrangeofyourmeasurements!
2. Contacts• [email protected]• [email protected]• [email protected]
MultidimensionalSmoothing
SaturationRepair
Sample1Sample2Sample3..
Rawdatafiles
Sample1Sample2Sample3..
IMPROVEDrawdatafiles
Pre-processing Processing
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3. Requirements• AcomputerrunningWindows7(64-bit)orlaterwithatleast8GBofRAM.• The.NETFramework4.0.• Agilent MassHunter data files produced by the 6560 Ion Mobility Q-TOF instrument when
operatedinthe“ionmobilitymode”.
4. Softwarefeatures• Single-clickbatchprocessingofmultipleIM-MSdatafiles.• IonmobilityMSwith/withoutanyseparation:LC-IM-MS,solidphaseextractionIM-MSanddirect
infusionIM-MS.• All-Ions data files (Data-Independent Acquisition) with alternating high/low collision energy
fragmentation.
PNNLPreProcessor|IonmobilityMS–USERGUIDE 3
5. UsageInstallationisnotrequired.Downloadandunzipthefileinyourcomputer.
• Doubleclickthe“PNNL-PreProcessor.exe”filetostartit.• Clickthetextboxtosearchandselectasingledatafile(.dfolder)oradirectorywhichcontains
severaldatafiles(.dfolders).• Adjusttheparametersandclickthe“Process”button.
For each data file, the progress bar will indicate the processing status and a new data file will begenerated.
IfyouusePNNLPreProcessor,pleasecite:
Bilbaoetal.comingsoon…
PNNLPreProcessor|IonmobilityMS–USERGUIDE 4
6. SettingparametersRawintensityvaluesineachframe(anIMseparation)aresmoothedfirstinthedriftdimensionfollowedbysmoothingofthechromatographic(orinfusion)dimensionconsideringneighboringframes.
6.1 NumberofpointsThenumberofpointsusedtocomputetheaverageornewsmoothedvalueshouldbespecifiedforeachdimension.Forinstance,using5pointsfordriftmeanthateachnewpointiscomputedastheaverageconsideringalsothe2driftspectrabeforeandthe2driftspectraafter(samem/zandwithinthesameframe).
• Asageneralrule,useanumberofpointsnolargerthanhalfofthepeakwidthatbase.Smoothingwithalargenumberofpointscauseadetrimentallostinresolution(seesectionbelowonOverSmoothing).
• Smoothing can be applied to allm/z, drift and chromatographic/infusion dimensions or anysubsetcombination.
• Smoothingacrossthem/zdimensionistypicallynotnecessaryforAgilentIM-MSsystems.• Largerchromatographic/infusionnumberofpointsrequiresmorememoryandprocessingtime.• For direct infusion data, the option “sum frames” can be selected as the number of
chromatographic/infusionpointstogenerateasingleframefile.Allframeswillbesummedpriorsmoothing. Inaddition,atextboxwillappear,whereasubsetof framescanbespecifiedasarangeaccordingtotheframenumber.Forinstance,using“10–30”,willfilteroutorignorethesignalinthefirst9framesandalltheframesaftertheframenumber30.Inthecaseoflow/highfragmentationdata, framesare summed independently foreach fragmentationenergyandasubrangereferstothenumberofframesofeachmodeindependently(i.e.,theframenumberobservedintheIM-MSBrowsershouldbedividedby2).Allframesaresummedwhenonlythesymbol“–”iskeptinthetextbox.
6.2 SignalintensitylowerthresholdAfterapplyingthesmoothing,intensityvaluesthatdonotexceedthethresholdwillnotberecordedinthefile.Whenusingtheoption“sumframes”,alsousealargerintensitythreshold(i.e.,multipliedbythenumberofframessummed).
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7. OverSmoothingWhenfirstusingthesoftwareorprocessingdatafromanewsampletype,youshouldcheckyourrawdatatoinspecttheresults.Oneofthepossibleproblemsthatyoumayrunintoisoversmoothing,orblurringtwopeaksintoone.Thishappenswhensmoothingisappliedwithanumberofpointsthatistoolarge(i.e.smoothtoomanypoints,relativetothewidthofapeak).
Toavoidoversmoothing,pleasedothefollowing:checkoneofyourfilesandseehowmanypointsyouhaveacrosstheearlydriftpeaksthatyouareinterestedin,thelowm/zrangehasusuallynarrowerdriftpeaksandearlierdrifttimes.
IntheIM-MSBrowser,select:Configuration->SetDriftSpectrumunitstoDriftBins.
Inthefollowingexample,thefileintheleftistheoriginalorunsmoothedone,thosedriftpeakshavelessthan~6pointsperpeak(thefirstonefrom158-164DriftBin),thosearelikelyisomers.Topreservethem,useonly3pointsforsmoothing(asinthemiddlefile),sinceusing5or7pointswillmergethosedriftpeaks(asintherightfile).
UNSMOOTHED 3pointsdrift 5pointsdrift
