Agilent G3835AA MassHunter Mass Profiler Professional Software Familiarization Guide

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1 Agilent G3835AA MssHunter Mss Profiler Professionl Softwre Fmiliriztion Guide Fmiliriztion Tutoril: How do I do new nlysis? 2 Advnced Tsks 25 Wht is Agilent Mss Profiler Professionl? Agilent Mss Profiler Professionl (MPP) softwre is powerful chemometrics pltform designed to exploit the high informtion content of mss spectr (MS) dt nd cn e used in ny MS-sed differentil nlysis to determine reltionships mong two or more smple groups nd vriles. MPP provides dvnced sttisticl nlysis nd visuliztion tools for GC/MS, LC/MS, CE/MS, ICP-MS, nd NMR dt nlysis. MPP lso integrtes smoothly with Agilent MssHunter Worksttion, Spectrum Mill nd ChemSttion softwre nd is the only pltform tht provides integrted identifiction/ nnottion of compounds nd integrted pthwy nlysis for metolomic nd proteomic studies. The system lso enles Automted Smple Clss Prediction tht revolutionizes mss spectrometer-sed qulittive nlysis of unknown smples in mny pplictions. MPP is idelly suited for pplictions chrcterized y complex smple mtrices such s metolomics, proteomics, nturl products, food, everges, flvors, frgrnces, nd environmentl nlyses. How do I get strted with Mss Profiler Professionl? MPP comes preloded with demonstrtion experiment to show you the functionlity of the product. A project clled Mlri contins n experiment clled Mlri LCMS ESI+ ph 7. You re encourged to explore this demonstrtion project to get to know Mss Profiler Professionl. Use the fmiliriztion tutoril to crete your first project nd experiment with the softwre. Agilent Technologies

2 Fmiliriztion Tutoril: How do I do new nlysis? The steps of the fmiliriztion tutoril re (1) Crete new project nd experiment for your dt, (2) Import nd orgnize your dt, (3) Perform your initil differentil nlysis, (4) Use MssHunter ID Browser to identify your compounds, nd (5) Sve your project Exercise 1. Crete new project nd experiment for your dt An esy wy to strt using the MPP softwre is to recrete the smple experiment from the demonstrtion project. The originl Mlri Demo dt you need is provided with the MPP instlltion. The dt files re locted in nmed folders within the min instlltion folder, usully found in C:\Progrm Files\Agilent\ MssHunter\Worksttion\Mss Profiler Professionl\smples (Windows 7). A new project nd experiment is creted through four sequentil dilog oxes: (1) Strtup, (2) Crete New Project, (3) Experiment Selection, nd (4) New Experiment. Follow the steps elow to crete new experiment with the Mlri Demo dt. 1 Strt Mss Profiler Professionl. 2 In the Strtup dilog ox, open recent project or crete new project. If you wnt to use the Mlri project: Click Open recent project. Select Mlri from the Select recent project list. c Click OK. The Strtup dilog ox helps you quickly set up new project or continue your nlysis with previous project. Open recent project opens the project nd experiment(s) tht re stored in the project. A project is continer for collection of experiments; ech experiment contins your smples, interprettions, nd nlyses. A project cn hve multiple experiments on different smple types nd orgnisms. 2 MPP Fmiliriztion Guide

3 d Select n opertion from the Workflow Browser to continue nlyzing the Mlri Demo project. The Workflow Browser is found long the right sider nd is orgnized in groups of opertions. If you insted wnt to crete new project, click Crete new project nd go to Step 3, otherwise proceed to Exercise 4 on pge In the Crete New Project dilog ox, enter your project informtion. c Type Mlri Demo Fmiliriztion in Nme. Type descriptive informtion in Notes. Click OK. The project nme nd notes my e viewed nd edited t ny time using the Project Inspector y clicking Project > Inspect Project from the menu r. MPP Fmiliriztion Guide 3

4 4 In the Experiment Selection Dilog dilog ox, crete new experiment. Click Crete new experiment. Click OK. If you click Open existing experiment, you re prompted for the experiment to dd to the project. This is the sme s clicking the Add experiment utton. 5 In the New Experiment dilog ox, enter nd select informtion tht guides your experiment cretion. c d e f Type descriptive nme for the experiment in Experiment nme. Select Mss Profiler Professionl for Anlysis type. Select Unidentified for the Experiment type. Select Anlysis: Significnce Testing nd Fold Chnge for Workflow type. Type descriptive informtion in Experiment notes. Click OK. You my lso crete new experiment from the Menu r, click Project > New Experiment, or from the Toolr, click the New experiment utton. Experiment type determines how Mss Profiler Professionl mnges the dt. You select Unidentified when the compounds hve only een identified y their moleculr fetures of neutrl mss nd retention time. You select Identified when the compounds hve een identified y compound, formul, nd/or CAS numer. You select Comined (Identified + Unidentified) when you re unsure if the dt hs een identified in full or in prt, or when MssHunter Qulittive Anlysis hs een previously used to identify some of the compound fetures. 4 MPP Fmiliriztion Guide

5 Exercise 2. Import nd orgnize your dt After you set up project nd crete n experiment, the MS Experiment Cretion Wizrd immeditely guides you through the necessry steps to orgnize your experiment, import your dt, define your experiment vriles, nd prepre your dt for nlysis. The MS Experiment Cretion Wizrd flow digrm is illustrted in Figure 1. The dt preprtion includes grouping, filtering, lignment, normlizing, nd selining. Figure 1 MS Experiment Cretion Wizrd 1 Select the dt source in the MS Experiment Cretion Wizrd (Step 1 of 11). Click MssHunter Qul nd select Homo spiens for the Orgnism if you re using the Mlri Demo dt set. Click Next. If you re using your own dt set, click the source of your smple files, nd select the Orgnism of the smple files or select None. Note tht selecting n Orgnism is most importnt when you use the Pthwy Anlysis fetures of MPP. MPP Fmiliriztion Guide 5

6 2 Select the smple dt to import in the MS Experiment Cretion Wizrd (Step 2 of 11). c d Click Select Dt Files to disply the file selection dilog ox. Select ll of the.cef files you wnt to import into the experiment. The exmple Mlri dt files re: 1-1_pH7_pos_01.cef 1-2_pH7_pos_01.cef 1-3_pH7_pos_01.cef 1-4_pH7_pos_01.cef 3-1_pH7_pos_01.cef 3-2_pH7_pos_01.cef 3-3_pH7_pos_01.cef 3-4_pH7_pos_01.cef Click Open to lod the selected files. Click Next. Note: The Open dilog likely lredy points to the smples directory in the min Mss Profiler Professionl instlltion directory, ut if not, rowse to C:\Progrm Files\Agilent\MssHunter\ Worksttion\Mss Profiler Professionl\smples\Mlri Demo. A progress indictor is displyed while the files re eing loded into MPP. 3 Review nd order the selected files tht you re importing in the MS Experiment Cretion Wizrd (Step 5 of 11). Review the order of your smples. The order does not need to e chnged unless n entry is out of sequence. Click Next. Note: This step presents you with the only opportunity you hve to reorder your smples. The smple order shown in this step is used in susequent views where the results re orgnized y smple. 6 MPP Fmiliriztion Guide

7 4 Define the smple grouping with respect to the independent vriles nd the replicte structure of your experiment in the MS Experiment Cretion Wizrd (Step 6 of 11). Click Add Prmeter. In Experiment Grouping, smples with the sme prmeter vlues re treted s replictes. For the Mlri Demo smples you specify which files contin dt from infected smples nd which contin dt from uninfected smples. An independent vrile is n essentil element, constituent, ttriute, or qulity in dt set tht is deliertely controlled in your experiment. An independent vrile is referred to s prmeter nd is ssigned prmeter nme. The ttriute vlues within n independent vrile re referred to s prmeter vlues. Smples with the sme prmeter vlue. c d Type nme for your Prmeter nme in the Add/Edit Experiment Prmeter dilog ox. Type Infection for the Mlri Demo. Click your replicte Smples tht shre the sme first prmeter vlue in your dt. For exmple: 1-1_pH7_pos_01 1-2_pH7_pos_01 1-3_pH7_pos_01 1-4_pH7_pos_01 Select the Prmeter type for your grouping. Non-Numeric is selected for the Mlri Demo. Prmeter Type options: Select Non-Numeric if the grouping is not quntittive vlue. Select Numeric if the grouping vlue is quntittive or vlue tht reflects degree of proportionlity mong the smples with respect to n independent vrile. A numeric prmeter type llows some dt plots to e scled y the prmeter vlues. e Click Assign Vlue. MPP Fmiliriztion Guide 7

8 f g h i Type the vlue for your first grouping in the Assign Vlue dilog ox. For the Mlri Demo type Not Infected. Click OK. Click your replicte Smples tht shre the sme second prmeter vlue in your dt. For exmple: 3-1_pH7_pos_01 3-2_pH7_pos_01 3-3_pH7_pos_01 3-4_pH7_pos_01 Click Assign Vlue. In the Mlri Demo exmple the smples re ssigned prmeter vlues representing the Infection prmeter nme. The highlighted smples re ssigned the vlue typed in the Assign Vlue dilog ox. You my chnge the vlue of ny smple, or group of smples; highlight the smple nd click Assign Vlue or Cler. j k Type the vlue for your second grouping in the Assign Vlue dilog ox. For the Mlri dt type Infected. Click OK. l Review your entries nd grouping ssignment ccurcy in the Add/Edit Experiment Prmeter dilog ox. m Click OK when the grouping for this prmeter nme is complete. 8 MPP Fmiliriztion Guide

9 n Click Next when you hve completed your experiment grouping. You cn sve your experiment grouping in t seprted vlue (TSV) file y using the Sve experiment prmeters to file utton. You cn view, edit, nd crete TSV files using Microsoft Excel or Windows Notepd. By using the Lod experiment prmeters from file utton you cn quickly pply previously creted or sved experiment grouping. Note: You my lso dd grouping or chnge your grouping during the Anlysis: Significnce Testing nd Fold Chnge Wizrd nd t ny time therefter. MPP Fmiliriztion Guide 9

10 5 Select nd enter the dt filter prmeters in the MS Experiment Cretion Wizrd (Step 7 of 11). c d e f g h Mrk the Minimum solute undnce check ox under Aundnce filtering. Type vlue of 5000 counts. Cler the Limit to the lrgest nd Minimum reltive undnce check oxes. Mrk the Use ll ville dt check ox under Retention time filtering. Cler the Use ll ville dt check ox nd type 50 for Min Mss, nd type 1000 for Mx Mss under Mss filtering. Click Minimum numer of ions nd type 2 for Minimum numer of ions under Numer of ions. Click Multiple chrge sttes foridden under Chrge sttes. Click Next. Filtering during the dt import process my e used to reject low-intensity dt or restrict the mss spectrl rnge of your dt. In Find Compounds y Formul (FF) generted dt files the term undnce ctully refers to the feture volume. Wheres, in Find Compounds y Moleculr Feture (MFE) generted dt files the term undnce ctully refers to the feture chromtogrphic re. Filtering y mss my improve your sttisticl nlysis y rejecting msses tht re not significnt to the experiment. Filtering works with oth GC/MS nd LC/MS dt. 10 MPP Fmiliriztion Guide

11 6 Select nd enter the retention time nd mss lignment prmeters in the MS Experiment Cretion Wizrd (Step 8 of 11). c d Cler the Perform RT Correction check ox under Retention time Correction. Type 0.1 % nd 0.15 min for RT Window under Compound lignment. Type 5.0 ppm nd 2.0 md for Mss Window. Click Next. Alignment prmeters re used y MPP to lign nd group compounds y similr mss nd retention time cross multiple smple files. The vlues you enter depend on your experimentl conditions nd the resolution of your MS instrument. In generl, lrger retention time shift my e used to compenste for less thn idel chromtogrphy nd smller mss window my e used for higher resolution mss spectrometers. It is recommended to inspect the qulity of your smple dt to determine the est vlues for your lignment prmeters. MPP Fmiliriztion Guide 11

12 7 View nd review the compounds present nd sent in ech smple in the MS Experiment Cretion Wizrd (Step 9 of 11). Cler the Export For Recursion check ox. Click Next. The unidentified compounds re ligned, nd MPP displys summry dt from the lignment. The Compound Frequency chrt nd tle report the numer of common entities tht pper in your smples (i.e., there re 474 entities tht pper in ll 8 smples, nd 1283 entities tht pper in only 1 smple - one-hit wonders ). The percent columns show you undnce distriution of the identicl entities normlized to the most undnt common entity. If most of the one-hit wonders hve low reltive undnce your smple dt lignment is likely good. If the one-hit wonders hve high reltive undnce (i.e., in the % column) then you my need to improve your smple dt lignment. In the Mss vs. RT tle, replicte smples re expected to hve similr numer of compounds present nd sent. Any prt of the smple summry my e exported; right-click on tht prt of the summry. Click Export As, nd click the imge type. The option Imge llows you to enter the imge file loction, file nme, imge size, nd resolution to meet your puliction requirements. 12 MPP Fmiliriztion Guide

13 8 Select whether to normlize the dt to reduce the vriility cused y smple preprtion nd instrument response in the MS Experiment Cretion Wizrd (Step 10 of 11). c Select None for the Normliztion Algorithm in the Normliztion t. Cler the Use Externl Sclr check ox in the Externl Sclr t. Click Next. You my use normliztion nd externl sclr techniques to reduce the vriility cused y smple preprtion nd instrument response in your dt. MPP Fmiliriztion Guide 13

14 9 Select whether to compre fetures in ech smple to the response of the fetures cross multiple smples in the MS Experiment Cretion Wizrd (Step 11 of 11). Click Bseline to of ll smples. Select medin for the Bseline to of ll smples. Click Finish. There re four selining options: None: Recommended if only few fetures in the smples exist. Z-Trnsform: Recommended if the dt sets re very dense, i.e., with dt where very few instnces of compounds re sent from ny smple, such s quntittion dt set from recursion. Bseline to of ll smples: The undnce for ech compound is normlized to its selected sttisticl undnce cross ll of the smples. This hs the effect of reducing the weight of very lrge nd very smll compound fetures on lter sttisticl nlyses. Bseline to of control smples: The undnce for ech compound is normlized to its selected sttisticl undnce cross just the smples selected s the control smples. This hs the effect of weighting the compound fetures to known vlue tht is considered to e norml in the popultion while reducing the effect of lrge nd smll compound fetures. 10 Continue to improve the qulity of your results nd produce n initil differentil expression for your nlysis. Becuse you chose Anlysis: Significnce Testing nd Fold Chnge for Workflow type in the New Experiment dilog ox you re immeditely directed to the next exercise. If you chose Dt Import Wizrd for Workflow type in the New Experiment dilog ox you re done creting your experiment nd cn use the opertions ville in the Workflow Browser. 14 MPP Fmiliriztion Guide

15 Exercise 3. Perform your initil differentil nlysis The Significnce Testing nd Fold Chnge Wizrd guides you through the steps necessry to enter prmeters nd vlues tht improve the qulity of your results nd produce n initil differentil expression for your nlysis. The Significnce Testing nd Fold Chnge Wizrd flow digrm is illustrted in Figure 2. Figure 2 Anlysis: Significnce Testing nd Fold Chnge Wizrd 1 Summry Report (Step 1 of 8): Review your dt in the Summry Report workflow step. Verify tht you see the Anlysis: Significnce Testing nd Fold Chnge Wizrd s shown elow. Click Next. The Anlysis: Significnce Testing nd Fold Chnge Wizrd shows profile plot nd series of steps to finish the experiment setup nd your initil nlysis. The current step is highlighted. You do not enter nlysis prmeters t this time. You my review the dt, chnge the plot view, export selected dt, or export the plot to file y click or right-click t desired fetures ville on the plot. MPP Fmiliriztion Guide 15

16 2 Experiment Grouping (Step 2 of 8): Enter the smple grouping with respect to the independent vriles nd the replicte structure of your experiment in the Experiment Grouping workflow step. Verify tht your Experiment Grouping looks like the exmple elow. Click Next. In Experiment Grouping, smples with the sme prmeter vlues re treted s replictes. In the Mlri Demo smples you specify which files contin dt from infected smples nd which contin dt from uninfected smples. If you re using your own dt set, click Add Prmeter nd set up prmeter nd enter the prmeter vlues for ech dt file nd return to this disply. 3 Filter Flgs (Step 3 of 8): Enter vlues tht filter entities in your smples sed on the qulity of their presence in specified smples nd conditions in the Filter Flgs workflow step. c d e f Review your dt, chnge the plot view, export selected dt, or export the plot to file. Click Re-run Filter to enter prmeters in the Filter Prmeters dilog ox. Mrk the Present nd Mrginl check oxes. Cler the Asent check ox. Click t lest out of X smples hve cceptle vlues nd type 2 in the entry ox. By setting this prmeter to vlue of two or more, one-hit wonders re filtered. Click OK. A flg is used to denote the qulity of n entity within smple. A flg indictes if the entity ws detected in ech smple s follows: Present mens the entity ws detected, Asent mens the entity ws not detected, nd Mrginl mens the signl for the entity ws sturted. This filter removes irreproducile entities from further considertion s you continue your nlysis. A mjor ojective of Filter Flgs is to remove one-hit wonders from further considertion. A one-hit wonder is n entity tht ppers in only one smple, is sent from the replicte smples, nd does not provide ny utility for sttisticl nlysis. 16 MPP Fmiliriztion Guide

17 g h Review the profile plot nd repet the Re-run Filter until you otin the est results for your experiment. Click Next. 4 Filter y Frequency (Step 4 of 8): Enter vlues tht filter the remining entities in your smples sed on their frequency of occurrence mong the smples nd conditions in the Filter y Frequency workflow step. c d e Review your dt, chnge the plot view, export selected dt, or export the plot to file. Click Re-run Filter to enter prmeters in the Filter Prmeters dilog ox. Type 100 in the Retin entities tht pper in t lest ox. Click of smples in t lest one condition. Click OK. Filter Frequency llows you to filter the input dt sed upon the frequency with which ny compound ppers in ech smple in the experiment. The filter is specified y typing the minimum percentge nd selecting the pplicle condition. MPP Fmiliriztion Guide 17

18 f g Review the profile plot nd repet the Re-run Filter until you otin the est results for your experiment. Click Next. 5 QC on smples (Step 5 of 8): Assess the smple qulity of your experiment using the QC on smples workflow step. Review your dt, chnge the plot view, export selected dt, or export the plot to file. Click Next. 18 MPP Fmiliriztion Guide

19 6 Significnce Anlysis (Step 6 of 8): Assess the differentil significnce nd fold chnge of your smples using the Significnce Anlysis workflow step. c d Review your dt, chnge the plot view, export selected dt, or export the plot to file. Move the p-vlue cut-off slider or type vlue to mke djustments. The defult vlue is Move the Fold chnge cut-off slider or type vlue to mke djustments. The defult vlue is 2.0. Click Next. In Significnce Anlysis, entities re filtered sed on their p-vlues clculted from sttisticl nlysis. The entities tht stisfy the Significnce Anlysis re pssed on for the Fold Chnge Anlysis. MPP shows the fold chnge of the undnce vlue for ech compound cross the vrious smples in the experiment. Set the fold chnge filter so tht you focus on the compounds tht hve intensity vlues tht re most significnt mong the smples in the experiment. The Significnce Anlysis wizrd pge llows you to djust the cutoff vlues for either the p-vlue or fold chnge. 7 Significnce Anlysis (Step 7 of 8): Becuse Fold Chnge ws included in the prior step (Step 6 of 8), this seprte Fold Chnge step is skipped. Fold Chnge is included in Step 6 of 11 ecuse the significnce nlysis is t-test due to the dt consisting of one prmeter with two conditions (prmeter vlues). MPP Fmiliriztion Guide 19

20 8 Significnce Anlysis (Step 8 of 8): Identify the entities in your experiment using the IDBrowser Identifiction workflow step. c Review the tle of compounds tht remin fter ll of the previous filtering steps. Click IDBrowser Identifiction. Continue with the next exercise. The compounds remining re lso referred to s entities. The compounds re identified only y their mss nd retention time in the Compound column of the tle. ID Browser helps you identify the compounds in the tle. 20 MPP Fmiliriztion Guide

21 Exercise 4. Use MssHunter ID Browser to identify your compounds Identify the compounds tht remin fter pplying the filtering steps of the Significnce Testing nd Fold Chnge Wizrd. 1 Set up your identifiction method in the first dilog ox of the Compound Identifiction Wizrd. Review the identifiction method. Click Next. When you lunch ID Browser it receives the list of entities (lso referred to s compounds) from the lst step of the previous exercise. If you strted ID Browser from the Workflow Browser, click Run ID Browser on the ID Browser toolr. 2 Set the prmeters for your identifiction technique in the second dilog ox of the Compound Identifiction Wizrd. Review the identifiction techniques. Click Finish. ID Browser provides you with mny wys to djust the identifiction lgorithm. Click Help to get full description of the options nd fetures ville to set up your identifiction technique. MPP Fmiliriztion Guide 21

22 3 Review nd djust your identifictions. Review the identifiction technique. Click Finish. ID Browser shows you informtion out ech identified entity nd gives you the ility to mke djustments to the identifictions. 4 Accept the current identifictions nd return to Mss Profiler Professionl. Click Sve nd Return on the ID Browser toolr. ID Browser sends ll of the informtion it hs generted ck to your entity list in Mss Profiler Professionl. 22 MPP Fmiliriztion Guide

23 5 Exmine the results in Mss Profiler Professionl. Study the compounds tht re now identified in the compound tle. Click Finish. The compound column in the tle shown in Significnce Anlysis (Step 8 of 8) is updted with your identifictions from ID Browser. 6 Review the profile plot, the Anlysis: Significnce Testing nd Fold Chnge Wizrd is now complete. Review the profile plot. Click on ny entity list in the Experiment Nvigtor to review the results. You cn follow the results from your nlysis in the hierrchicl presenttion in the Experiment Nvigtor on the left sider. MPP Fmiliriztion Guide 23

24 Exercise 5. Sve your project Sve your current nlysis s TAR file for rchiving, restortion of ny future nlysis to the current results, shring the dt with collortor, or shring the dt with Agilent customer support. 1 Export your project to TAR file. Click Project > Export Project. Mrk the check ox next to the experiment you wish to sve c Click OK. You hve completed creting your project nd nlyzing n experiment. It is recommended to rchive your progress y exporting your project. d e f Select or crete the file folder. Type your file nme in File nme. Click Sve. g Click OK. 24 MPP Fmiliriztion Guide

25 Advnced Tsks The opertions ville in the Workflow Browser provide the tools necessry for nlyzing fetures from your mss spectrometry dt depending upon the need nd im of the nlysis, the experiment design, nd the focus of the study. This helps you crete different interprettions to crry out the nlysis sed on the different filtering, normliztion, nd stndrd sttisticl methods. It is recommended tht you follow the procedures in the Mss Profiler Professionl Softwre - Appliction Guide for n dditionl level of detiled informtion to help you use MPP with your dt. The Metolomics Discovery Workflow nd Mss Profiler Professionl User Mnul provide you with techniques nd explntions to perform dvnced nlysis tsks. MPP Fmiliriztion Guide 25

26 BioCyc Pthwy/Genome Dtses Includes BioCyc Pthwy/Genome dtses from the Bioinformtics Reserch Group t SRI Interntionl, used under license. Cittion sed on use of BioCyc Users who pulish reserch results in scientific journls sed on use of dt from the EcoCyc Pthwy/Genome dtse should cite: Keseler et l, Nucleic Acids Reserch 39:D Users who pulish reserch results in scientific journls sed on use of dt from most other BioCyc Pthwy/Genome dtses should cite: Cspi et l, Nucleic Acids Reserch 40:D In some cses, BioCyc Pthwy/Genome dtses re descried y other specific pulictions tht cn e found y selecting the dtse nd then going to the Summry Sttistics pges under the Tools menu. The resulting pge sometimes contins cittion for tht dtse. 26 MPP Fmiliriztion Guide

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28 In this ook The Agilent G3835AA MssHunter Mss Profiler Professionl Softwre - Fmiliriztion Guide presents the functionlity of Mss Profiler Professionl using Mlri project contining n experiment clled Mlri LCMS ESI+ ph 7. Windows7 is registered trdemrk of Microsoft Corp. in the US nd/or other countries. Agilent Technologies, Inc Revision A, Novemer 2012 *G * G Agilent Technologies