Download - PNNL PreProcessor | Ion mobility MS - Integrative … PreProcessor | Ion mobility MS USER GUIDE – version 2017.08 In close collaboration between Pacific Northwest National Laboratory

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

PNNLPreProcessor|IonmobilityMS–USERGUIDE 2

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.


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…


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).


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