Home

Mass Profiler Pro Application_Guide F8.book

1. e Ifyou selected Analysis Significance Testing and Fold Change for the Workflow type in the New Experiment dialog box you immediately begin your analysis 22 MPP Application Guide 4 Create your Initial Analysis MPP Application Guide The Analysis Significance Testing and Fold Change Wizard Figure 4 improves the quality of your results and helps you create an initial differential expression from your data The steps are predetermined and based on the experiment type experiment grouping and conditions you entered when creating your project and setting up your experiment Some steps may be automatically skipped for your experiment Flow Chart of the Analysis Significance Testing and Fold Change Wizard te ee a ee Step 1 Step 4 Summary Report Filter by Frequency ere ess Step 8 ID Browser Identification V Step 2 Step 5 Experiment Quality Control on Grouping Samples Step 6 Filter Flags Analysis perform advanced tasks Step 3 Significance i Save your project and Go to Step4 Goto Step Figure 4 Analysis Significance Testing and Fold Change Wizard 23 Steps Detailed Instructions Comments 1 Review the summary of your new a Review the Summary Report e Familiarize yourself with the tools experiment Summary Report b Click and right click features on the available to you in the summary Step 1 of 8 plot or spreadsheet to review the report vi
2. e Menu bar Click Project gt New Experiment Toolbar Click the New experiment button itt Open existing experiment opens a project and the experiment s that are stored in the project You may also click the Add experiment button to add an existing experiment to your project Regardless of your personal expertise it is recommended to select the Analysis Significance Testing and Fold Change for the Workflow type to provide you with quality control to your analysis that improves your results At the conclusion of the Analysis Significance Testing and Fold Change workflow you may save your project and customize your entire analysis using the operations available in the Workflow Browser MPP Application Guide Steps Detailed Instructions Comments e Table 1 below and Table 2 on page 10 show the selection and entry options available to you for the New Experiment dialog box Experiment type see also Table 2 determines how Mass Profiler Professional manages the data i x gt Select Unidentified when the Experiment description compounds have only been identified by their molecular Enter a name analysis type experiment type and a desired workflow type Analysis will guide you through a statistical significance best and Fold change analysis Data Import will guide you through experiment creation features of neutral mass and ante ie Prediction will guide you through the creation and testing of a p
3. Click Next Comments Set the minimum and the applicable condition of samples that an entity must be present to pass the filter 1 of all samples conditions are not evaluated 2 of samples in only one condition one and only one condition 3 of samples in at least one condition one or more conditions and 4 of samples within each condition all conditions For experiments that contain five or fewer replicates 100 of all samples is recommended For experiments with a larger number of replicates the filter frequency percentage may be lowered A larger removes more entities Steps Filter By Frequency 1 Summary Report 2 Experiment Grouping Entities are filtered based on their Frequency of occurance across samples Define the stringency of the filter by selecting the minimum percentage of samples in which entity must pass the filter or by selecting the minimum percentage of samples within any x out of y conditions in which the entity must pass the filter To change the filter criteria click on the Re run Filter button 3 Filter Flags 4 Filter By Frequency 5 QC on samples 6 Significance Analysis 7 Fold Change 8 IDBrowser Identifica Normalized Intensity Values Infected Displaying 917 of 2107 entities where at least 100 0 percent of samples in any 1 out of 2 conditions has flag P Infection Help MPP Application Guide Not Infected lt lt
4. a Click Add Parameter Elms Experiment Creation Wizard Step 6 of 11 f 3 x Experiment Grouping values here Experiment parameters define the grouping or replicate structure of your experiment Enter experiment parameters by clicking on the Add Parameter button You may enter as many parameters as you like but only the first two parameters will be used for analysis in the guided workflow Other parameters can be used in the advanced analysis You can also edit and re order parameters and parameter Displaying 8 sample s with 0 experiment parameter s To change use the button controls below Seen Lb H _pos_O1 H _pos_O1 H _pos_O1 H _pos_O1 H _pos_O1 US US US US Re Re BW NR B BR H _pos_O1 P P P P _pH _pos_O1 P P P H7_pos_01 Add Parameter Edit Parameter Delete Parameter Help Eladd Edit Experiment Parameter l xj Grouping of Samples Samples with the same parameter values are treated as replicate samples To assign replicate samples their parameter values select the samples and click on the Assign Yalues button and enter the value For the group Set the parameter type to numeric to interpret the parameter values as numbers Parameter name infection Parameter type Non Numeric z Samples Parameter Values Assign Value Clear Cancel lt lt Back Next gt gt Finish Can
5. appropriate icon Parameter Values Cancel 6 OPTIONAL Saving and importing experiment grouping information in a spreadsheet c Re order the parameter values by selecting a parameter column then click the Re order parameter values TE button Click one or more values that you want to reorder Click the Up O or Down button to reorder the selected value s Click OK when the order for this parameter is complete Save the experiment parameters and parameter values to a tsv Click the Save experiment parameters to file button 354 Load your experiment parameter grouping values from a tsv file instead of using the MPP user interface Click the Load experiment parameters from file button 5 Comments When you have more than one parameter associated with your samples each parameter and Its values is displayed in a separate column in the MS Experiment Creation Wizard Step 6 of 11 dialog box When the parameter column is selected the column is highlighted An example experiment grouping file that is in the Malaria Demo directory named MALARIA EXPERIMENT PARAMETERS to be loaded from file tsv The tsv file is organized using tab separated values tsv that may be created edited and viewed using Microsoft Excel or Notepad MPP Application Guide 17 Steps Detailed Instructions Comments c Load your experiment parameter grouping values from a sample file
6. 20 ee Agilent G3835AA MassHunter 7 e Mass Profiler Professional Software Application Guide 1 Prepare for your Experiment 4 2 Find the Features in your Data 6 3 Import and Organize your Data 7 4 Create your Initial Analysis 23 5 Save your project 37 6 Perform Advanced Operations 38 What is Agilent Mass Profiler Professional Agilent Mass Profiler Professional MPP software is a powerful chemometrics platform designed to exploit the high information content of mass spectra MS data and can be used in any MS based differential analysis to determine relationships among two or more sample groups and variables MPP provides advanced statistical analysis and visualization tools for GC MS LC MS CE MS ICP MS and NMR data analysis MPP also integrates smoothly with Agilent MassHunter Workstation Spectrum Mill and ChemStation software and is the only platform that provides integrated identification annotation of compounds and integrated pathway analysis for metabolomic and proteomic studies The system also enables Automated Sample Class Prediction that revolutionizes mass spectrometer based qualitative analysis of unknown samples in many applications MPP is ideally suited for applications characterized by complex sample matrices such as metabolomics proteomics natural products food beverages flavors fragrances and environmental analyses aig Agilent Technologies Where is MPP used in your experiment MPP is u
7. Back Next gt gt Einish Cancel 27 Steps Detailed Instructions 5 Assess the sample quality of your a Review the summary plot experiment QC on samples Step b Highly recommended Click Back to make adjustments to prior steps in the 5 of 8 workflow to improve the results c Click Next el workflow Type Analysis Significance Testing and Fold Change Step 5 of 8 Comments QC on samples provides you with the first view of the data using a Principle Component Analysis PCA PCA allows you to assess the data by viewing a 3D scatter plot of the calculated principle components You want your samples to form discrete groups in the 3D PCA Scores view based on their parameter assignments Steps 1 Summary Report 2 Experiment Grouping 3 Filter Flags 4 Filter By Frequency 5 QC on samples 6 Significance Analysis 7 Fold Change QC on samples Sample quality can be assessed by examining the values in the PCA plot and other experiment specific quality plots 8 IDBrowser Identifica Displaying 8 out of 8 samples retained in the analysis H _pos_O1 Not Infected H7_pos_01 Not Infected 1 1_p 1 2_p 1 3_pH _pos_01 Not Infected 1 4_pH _pos_O01 Not Infected 3 1_pH _pos_01 Infected 3 2_pH _pos_O1 Infected 3 3_pH _pos_O1 Infected 3 4_pH _pos_O1 Infected gt ae _Z Axis X Axis at Tae ae Y Axis Componen
8. Details Detailed Instructions a lypeMalaria Project or your project information in Name b Type descriptive information in Notes c Click OK x Name Malaria Project Notes Project containing the Agilent Malaria Demol 4 inthe Experiment Selection Dialog dialog box create a new experiment Lox cancel_ a Click Create new experiment b Click OK lexperiment Selection Dialog Ea 4n experiment is an organized collection of LC MS or GC MS sample data from a given data source IF you have an experiment you wish to use from a previous project please choose Open existing experiment You may also create a new experiment with new data or previously imported data Choose Experiment C Open existing experiment Help 5 Inthe New Experiment dialog box enter and select information that guides your experiment creation Type a descriptive name for the experiment in Experiment name b Select Mass Profiler Professional for Analysis type c Select Unidentified or Combined Identified Unidentified for the Experiment type d Select Analysis Significance Testing and Fold Change for Workflow type e Type descriptive information in Experiment notes f Click OK Comments The project name and notes may be viewed and edited at any time using the Project Inspector by clicking Project gt Inspect Project from the menu bar You may also create a new experiment in your project from the
9. extracted by processing your data files with Agilent MassHunter Qualitative Analysis MPP imports and analyzes the features that are saved in your CEF files MassHunter Qualitative Analysis MassHunter Qualitative Analysis is used in conjunction with MassHunter DA Reprocessor to perform untargeted feature extraction and additionally with MPP to perform recursive targeted feature extraction Feature finding with MassHunter Qualitative Analysis involves performing the following steps 1 Create an untargeted Find by Molecular Feature MFE method in MassHunter Qualitative Analysis 2 Run the MFE method using DA Reprocessor to extract and save the untargeted features from the sample data files 3 Import align and filter the untargeted features using MPP 4 Export the features from MPP for targeted recursive finding in MassHunter Qualitative Analysis 5 Create a targeted Find by Formula FbF method in MassHunter Qualitative Analysis 6 Run the FbF method using DA Reprocessor to re extract and save the targeted features from the sample data files 6 MPP Application Guide 3 Import and Organize your Data Create a new project and experiment for your data You are guided through four sequential dialog boxes to create a new project and experiment to receive your data 1 Startup Select the option to create a new project 2 Create New Project Type descriptive information about your project 3 Experiment Selection Select
10. of a large number of dependent variables the features compounds that are measured in your samples The results must be significant beyond natural variability After you obtain your samples acquire your data and find the features in your sample data MPP takes you through data extraction processing and statistical analysis so that you can prove or disprove your hypothesis Elements to consider in planning your experiment The hypothesis The hypothesis is the question that is answered by your analysis For example the question may be a statement that proposes a possible correlation or cause and effect between a set of independent variables and the resulting features in your data Natural variability It is important to understand how any one sample in your data represents the population as a whole Because of natural variability and the uncertainties associated with both the measurement and the population no assurance exists that any single sample from a population represents the mean of the population Thus increasing the sample size greatly improves the accuracy of the sample set in describing the characteristics of the population 4 MPP Application Guide Replicate sampling Sampling the entire population is not typically feasible because of constraints imposed by time resources and finances On the other hand fewer samples increase the probability of making a false positive or false negative correlation System su
11. the option to create a new experiment as part of your project 4 New Experiment Set up the information to store with your experiment and to guide the analysis process Follow the steps below to setup your new project The Agilent Malaria Demo data set is used as an example in each step You are encouraged to substitute the demo information and data files with your own data Steps Detailed Instructions Comments 1 Start Mass Profiler Professional a Click the Mass Profiler Professional e When MPP starts if you choose icon on your desktop you are immediately guided through four sequential dialog boxes to create a new project and experiment 2 Create a new project from the a Click Create new project e Create new project provides you Startup dialog box b Click OK with the option to create a new experiment or import an experiment x from an existing project into the Welcome to MassProfiler Pro new project Select what you would like to do from the options below then dick on OK to continue Options e After closing an open project you Create new project may create a new project from the Z nti Menu bar click Project gt New i Project or from the Toolbar click SCIECGLTECENTDrOJEGE IEW FrOJECE the New project button E Do not show this dialog again e MPP Application Guide 7 Steps 3 Inthe Create New Project dialog box enter your project information Create New Project New Project
12. type you need to select sample files to import in the MS depends on the data source you Experiment Creation Wizard Step selected in the MS Experiment 2 of 11 Creation Wizard Step 1 of 11 x Select Data to Import See Table 2 on page 10 for a Data may be imported from files or previous experiments comprehen sive list of data sources you may select from based on your experiment type Selected files and samples e To control your progress through the wizard dialog boxes elect Data Files Select Samples Remove Help lt lt Back Next gt gt Finish Cancel b Select your samples in the Open dialog e Click Next gt gt to go to the next box If necessary browse to step C Program Files Agilent e Click lt lt Back to return to prior MassHunter Workstation Mass steps and make modifications to Profiler Professional samples your settings and previous Malaria Demo for the Malaria Demo entries c Click the sample molecular feature e Click Cancel to end the MS data files to import into the Experiment Creation Wizard experiment The example Malaria data without saving files are e 1 1 _pH _pos_01 cef e You may select a continuous range e 1 2_pH _pos_01 cef of files with a click on the first file e 1 3 _pH _pos_01 cef and press Shift and click on the last e 1 4 pH _pos_01 cef file that includes the range of files e 3 1 _pH _ pos 01 cef you want to select e 3 2 pH _pos_ 01 cef e 3
13. 13 N7 025 2 43E 11 218809 95 274 5602 0 62724996 2 26E 03 220 C19 H17 N3 017 52 3 56E 11 4975 7 84 c6 H5 NO 7 90E 12 214511 75 C16 H6 N2 0185 6 76E 11 42292 68 850 7883 0 74450004 9 57E 14 291330 66 C5 H6 N4 03 3 5 26E 12 3825816 50 C17 H9 Cl2 N 08 55 5 41E 12 307682 91 C23 H17 N3 014 3 31E 11 49340 76 C18 H9 N3 010 0 936 3 67E 11 490408 84 C28 H25 N 018 4 72E 09 2 52E 10 down 16 00 315417 03 18 27 C6 H6 N2 O 5 41E 05 3 90E 06 down 4 30 4 30 2 11 156 5247 2 66825 2 18E 10 4 29E 12 down 16 00 5 4384 98 15 73 C5 H11 N5 052 9 15E 11 1 40E 12 down 16 00 33049 86 15 01 C8 H14 N4 035 2 71E 09 1 15E 10 down 16 00 128892 02 16 98 11 UIA MD A enc 11 A EDC 1 dawn IE AN ADEO AA 10 A lt lt Back fext gt gt Finish Cancel e The Analysis Significance Testing and Fold Change workflow is now complete and you are immediately returned to the main MPP interface MPP Application Guide 35 Steps Detailed Instructions Comments bt Toe r zae L d i A o J J P p n y Bitton Project Navigator oo u peor wane at g a experiment organized into sequential mea gt cro ise ia ments z VUN T EAEN ajusa current project VA tee f pm z fio NN game he Aa io fe fee ARA j hui TNN Ni E eccoertes Yo AF eles Prd Hra Drima yo eion Threaten ho
14. 3 Compound Identification Browser Please select the identification methods you wish to apply to the compounds in this CEF file M Compound selection C Identify only highlighted compounds Identify only unidentified compounds Identify all compounds M Compound identification methods IV Database search CSV PCD METLIN ms pectral library search MV Molecular formula generator MFG Generate formulas for all compounds Generate formulas only for unidentified compounds Help Back Next gt gt Finish _ Cancel MPP Application Guide 31 Steps Detailed Instructions Comments f Setup the parameters and values for your database search El compound Identification Wizard b x Compound Identification Browser Please set parameters for identification techniques M Identification method C MassHunter Methods B 05 00 Defauit m l Identify Compounds oe ee amp d Search Database Generate Formulas e al C Mass and retention time retention time optional C Mass and retention time retention time required m Match tolerance Retention time fp 100 minutes El compound Identification Wizard Compound Identification Browser Please set parameters for identification techniques M Identification method C MassHunter Methods B 05 00 Default m r Search Criteria 3 Identify Compounds ereeeeeeeee B Search Da
15. 3 pH pos 01 cef e You may select discontinuous 3 4 pH pos 01 cef individual by pressing Ctrl and 2d clicking on additional files Look in rr Malaria Demo F d E El Recent Items File name pos_O1 cef 3 3_pH _pos_O1 cef 3 4 _pH _pos_O1 cef Open Files of type MassHunterQual Combined CEF v Cancel 12 MPP Application Guide Steps Detailed Instructions Comments d Click Open to load the selected files e Replicate samples are from the e Click Next collection of multiple identical samples from a population When x replicate samples are evaluated a iar a result is obtained that more closely ata may be imported from files or previous experiments approximates the true value of the 5 1 1_pH _pos_01 cef population 1 2_pH _pos_01 cef 1 3_pH _pos_0O1 cef F 1 4_pH7_pos_O1 cef e You can review and make changes Br1_pH7_pos D1 cof to your selection during the next 3 2_pH _pos_O1 cef i a 3 3_pH7_pos_01 cef step before finalizing the o e experiment creation i __ Select Samples Remove lt lt Back Next gt gt Finish Cancel Progress e A progress indicator is shown while We your files are imported into MPP 3 Review and order the sample files a Click one or more samples that you e Note This step is the only based on the independent want to reorder Opportunity to reorder your variables in your experiment inthe b Cli
16. EE i 1SE 0 28E 0 BR GVE 0 DOTE 0 TLO ra es ee Te ee i ey ee eed Fy GIA Jaep Eo LTTE FEED GISE O Ti A Le eLee 15 9604 ijito BE od ood ra Eo t i575 ETRIE Eao IOE LTE i oes a ttl la 4il nil IE GLN ETSO Aarel LITE ai ies ioa ir atal it Fiil T196 GEO LSE FAEG 65 obo dnai beroi FE EII ee i o a Fy aE TELH CHAO a Saf oh t iii cI ata lt The TREE ON LOJET hee ee ee oii a CE O Lak lt 0 74E 0 TA di isa LA a7 E F E F fiesob Liae The statistical analysis is either a T test or an Analysis of Variance ANOVA based on the samples and experiment grouping The last row of data in the Result Summary spreadsheet shows the number of entities that would be expected to meet the significance analysis by random chance based on the p value specified in each column heading If the number of entities expected by chance is much smaller than those based on the corrected p value your entities show significance among the parameter values The display of a diagram Venn Diagram Fold Change none or other plot depends on your samples and experiment grouping for the analysis MPP Application Guide 29 Steps Detailed Instructions Comments 7 Filter the remaining entities in your a Review the summary plot e The Fold Change workflow step samples based on their relative b Move the Fold change cut off slider or may be automatically skipped abundance ratios among the samples and conditions F
17. Molecular Feature MFE generated data file the term abundance actually refers to the feature volume In a Find by Formula FbF generated data file the term abundance actually refers to the feature chromatographic area 18 MPP Application Guide Steps Detailed Instructions d Mark the Use all available data check box under Retention time filtering e Clear the Use all available data check box and type 50 00 for the Min Mass and 1000 for the Max Mass under Mass filtering f Click the Minimum number of ions button and type 2 under Number of ions g Click Multiple charge states forbidden under Charge states h Click Next Clear the Perform RT correction check box Type 0 1 and 0 15 min for RT Window A smaller value reduces compound grouping and leads to a larger list of unique compounds c lType5 0ppmand 2 0 mDa for Mass Window It is not recommended to set the mass window less than 2 0 mDa for higher masses d Click Next Elms Experiment Creation Wizard Step 8 of 11 q x 8 Align the features across the a samples based on tolerances established by retention time and b mass in the MS Experiment Creation Wizard Step 8 of 11 Alignment Parameters Unidentified compounds from different samples are aligned or grouped together if their retention times are within the specified tolerance window and the mass spectral similarity as determined by a simple dot product calculation above the specifie
18. Next gt gt Finish Cancel a Clear the Export for Recursion check Comments This step shows a summary of the compounds present and absent in each of the samples based on the experiment parameters including the application of the filter and alignment parameters The Compound Frequency chart and table report the number of common entities that appear in your samples i e there are 474 entities that appear in all 8 samples and 1283 entities that appear in only 1 sample one hit wonders The percent columns show you abundance distribution of the identical entities normalized to the most abundant common entity If most of the one hit wonders have a low relative abundance your sample data alignment is likely good If the one hit wonders have a high relative abundance i e in the 30 100 column then you may need to improve your sample data alignment In the Mass vs RT table replicate samples are expected to have a similar number of compounds present and absent Use the Back and Next feature to independently assess the effects of your retention time alignment versus compound alignment It is not recommended to export the compounds for recursion at this step in your experiment Better results are obtained after the data has been filtered for significance MPP Application Guide Steps Detailed Instructions Comments 10 Select whether to normalize the a Select None for the Normalization e
19. You may use normalization and data to reduce the variability Algorithm external scalar techniques to caused by sample preparation and b Clear the Use External Scalar check reduce the variability in your data instrument response in the MS box that was caused by sample Experiment Creation Wizard Step c Click Next preparation and instrument 10 of 11 response x Normalization Criteria The compounds associated with each sample may be normalized to an internal standard percentile shift quantile and or an external scalar Normalization External Scalar Normalization Algorithm Internal Standard Percentile Shift Quantile None lt lt Back Next gt gt Finish Cancel Elms Experiment Creation Wizard Step 10 of 11 7 x Normalization Criteria The compounds associated with each sample may be normalized to an internal standard percentile shift quantile and or an external scalar Normalization External Scalar lt lt Back Next gt gt Finish Cancel MPP Application Guide 21 Steps Detailed Instructions Comments 11 Compare the features in each a Clickthe Baseline to _ ofall e There are four baselining options sample to the response of each samples button feature across multiple samples or b Select median for the Baseline to e None Recommended if only a few the control samples in the MS ____ of all samples fe
20. ameter values here Significance analysis step will be skipped if there are no replicates in any of the condition s Fold change analysis will be skipped if more than one parameter is entered and if the second parameter increases the number of conditions 4 Filter By Frequency 5 QC on samples 6 Significance Analysis 7 Fold Change Displaying 8 sample s with 1 experiment parameter s To change use the button controls below Not Infected H7_pos_01 Not Infected 8 IDBrowser Identifica 1 1_p 1 2_p 1 3_pH _pos_01 Not Infected 1 4_pH _pos_O01 Not Infected 3 1_pH _pos_O01 Infected 3 2_pH _pos_O1 Infected 3 3_pH _pos_O1 Infected 3 4_pH _pos_O1 Infected e 3 Filter entities from your samples based on the quality of their presence in specified samples and conditions Filter Flags Step 3 of 8 Add Parameter Edit Parameter Review the summary plot Click Re run Filter to enter parameters into the Filter Parameters dialog box Mark the Present and Marginal check boxes aG Parameters xj Acceptable Flags JV Present JV Marginal I Absent Retain Entities in which C atleast 100 0 of the values in any 1 out of 2 conditions have acceptable values atleast 2 out of 8 samples have acceptable values Cancel Delete Parameter lt lt Back Next gt gt Finish Cancel A flag is a term used to denote
21. atures in the samples exist Experiment Creation Wizard Step c Click the Finish button lt lt 8e 11 of 11 e 2Z Transform Recommended if the data sets are very dense data where very few instances of compounds are absent from any sample such as a quantitation data set from recursion e Baseline to of all samples The abundance for each compound is normalized to its selected x statistical abundance across all of Baselining Options the samples This has the effect of There are four baseline options reducing the weight of very large and very small compound features None This option will treat compounds with large intensities as more significant than compounds with lesser intensities 2 Transform This option should be used when comparing data from different sources lo ty inner ero eu ele x EEE SE E piv Quay vagarebaa on later statistical analyses their intensity Options e Baseline to of control C None samples The abundance for each C 2 Transform compound is normalized to its Baseline to median of all samples selected statistical abundance Baseline to meden gt of control samples across just the samples selected as index Samptes Control Samples the control samples This has the ae effect of weighting the compound features to a known value that is considered to be normal in the population while reducing the effect of large and small compound a cel features
22. cel Type a name for your Parameter name in the Add Edit Experiment Parameter dialog box Type Infection forthe Malaria Demo Click your replicate Samples that share the same first parameter value in your data For example e 1 1_pH7_pos_01 e 1 2 pH7_pos_01 e 1 3 pH7_pos_01 e 1 4 pH7_pos_01 Select the Parameter type for your grouping Non Numeric is selected for the Malaria Demo Click Assign Value Comments Note Grouping at this time is optional You may add grouping or change your grouping during the Analysis Significance Testing and Fold Change Wizard or at any time thereafter An independent variable is an essential element constituent attribute or quality in a data set that is deliberately controlled in your experiment An independent variable is referred to as a parameter and is assigned a parameter name The attribute values within an independent variable are referred to as parameter values Samples with the same parameter value and the same parameter name are treated as replicates Parameter Type options Select Non Numeric if the grouping is not a quantitative value Select Numeric if the grouping value is quantitative or a value that reflects a degree of proportionality among the samples with respect to an independent variable A numeric parameter type allows some data plots to be scaled by the parameter values 14 MPP Application Guide Steps Assign Yalue x Enter a
23. ck the Up le or Down button samples After completing the data MS Experiment Creation Wizard to reorder the selected sample s import create a new project or Step 5 of 11 c Repeat the reordering actions as often experiment and repeat this process as necessary to obtain your order to reorder your samples d Mark the sample names that you want to import into your experiment e You may select a continuous range e Click Next of files with a click on a first file and a Shift click on a last file that Els Experiment Creation wizara Step sora S a a a Sample Reordering to select To re order the samples select the samples and use the appropriate buttons on the right to move samples up or down This sample order will be used throught out the experiment a Deselect the samples that need not be imported Click the Restore al button at any select Sample time to return the sample order to Iv 1 1_pH _pos_01 i your starting point when this step 1 3_pH7_pos_01 1 4_pH7 _pos_01 was begun 3 1_pH7_pos_01 3 2_pH7_pos_01 3 3_pH _pos_O1 3 4_pH _pos_01 lt 1 lt 1 lt lt lt 1 lt lt Select All Unselect All lt lt Back Next gt gt Finish Cancel MPP Application Guide 13 Steps 4 Define the sample grouping with respect to the independent variables and the replicate structure of your experiment in the MS Experiment Creation Wizard Step 6 of 11 Detailed Instructions
24. d level f Retention time Correction J Perform RT correction Maximum Allowed RT Shirt fe us Yo fos min Mass Window fis 0 ppm 4 o Da RT Correction Method Without St f Cy t fio of Internal Standards E RTiminuUtesS Compound alignment RT Window fo 1 Yo jo 1s min Mass Window 5 0 ppm 2 0 mDa lt lt Back Next gt gt Finish Cancel Help Comments Filtering by maximum mass may improve your statistical analysis by rejecting masses that are not significant to the experiment This is especially relevant to metabolomic samples The filter parameters may be cleared to preserve the prior filtering that was used to generate the feature data file Filtering works with both GC MS and LC MS data This step is omitted when the experiment type is identified GC MS data alignment includes retention time difference and mass spectral match factor A large retention time shift may be used to compensate for less than ideal chromatography If retention time correction is used it is recommended to use at least two widely spaced standards and to use standards that are present in every sample The correction Is based on a piecewise linear fit Unidentified compounds from different samples are aligned or grouped together if 1 their retention times are within the specified tolerance window and 2 the mass spectral similarity are above the specified level Retention alignment rewrit
25. ed ae ai eid OPTIONAL Saving and importing time thereafter Sen ET experiment grouping information in a 3 3_pH7_pos_01 Infected A 3 4_pH7 pos 01 Infected spreadsheet These steps provide advanced instructions to manage your parameters and parameter name assignments using the wizard toolbar and a spreadsheet application Gear Cancel 4ssign value q Click Next when you have completed your experiment grouping Elms Experiment Creation Wizard Step 6 of 11 x Experiment Grouping Experiment parameters define the grouping or replicate structure of your experiment Enter experiment parameters by clicking on the Add Parameter button You may enter as many parameters as you like but only the first two parameters will be used for analysis in the guided workflow Other parameters can be used in the advanced analysis You can also edit and re order parameters and parameter values here Displaying 8 sample s with 1 experiment parameter s To change use the button controls below 2h BEE 1 1_pH _pos_01 Not Infected 1 2_pH _pos_01 Not Infected 1 3_pH _pos_01 Not Infected 1 4_pH _pos_01 Not Infected 3 1_pH _pos_01 Infected 3 2_pH _pos_O1 Infected 3 3_pH _pos_O1 Infected 3 4_pH _pos_01 Infected Edit Parameter Delete Parameter lt lt Back Next gt gt Finish Cancel 16 MPP Application Guide Steps 5 OPTIONAL Re order yo
26. ents for individual or multiple samples as necessary to make corrections or changes Click OK when the grouping for this parameter name is complete Comments In this example the samples are assigned parameter values representing the Infection parameter The highlighted samples are assigned the value typed in the Assign Value dialog box MPP Application Guide 15 Steps Detailed Instructions Comments p Repeat Add Parameter if yourdatahas You may change the value of any more than one independent variable sample or group of samples Bl Add edit Experinent Ea T x Click Add Parameter highlight the sample and click Grouping of Samples n Samples with the same parameter values are treated as replicate Repeat the steps above until you Assign Value or Clear samples To assign replicate samples their parameter values select z ign Values have assigned a parameter name type and value to all of your data s the samples and click on the Assign Yalues button and enter the value For the group Set the parameter type to numeric to interpret the parameter values as numbers Parameter name Infection Parameter type Non Mumeric Note You may add grouping or change your grouping during the Analysis Significance Testing and Fold Change Wizard and at any e Review step 5 OPTIONAL Re order your parameter values and step 6 Sames O Parameter Values Not Infected 1 2_pH _pos_01 Not Infect
27. es the retention times in the data file MPP Application Guide 19 Steps Detailed Instructions 9 Review the compounds present and absent in each sample in the box MS Experiment Creation Wizard b Click Next Step 9 of 11 Elms Experiment Creation Wizard Step 9 of 11 j x Sample Summary Hovering the mouse over a compound in the graph will reveal the complete identity of the compound 4 right click mouse action on the graph or the spreadsheet will offer additional display and export options Total number of Aligned Compounds 3390 Mass Vs RT Compound Frequency Total Samples 8 1200 1000 800 600 No of Compounds TEREE Frequency Frequency Number 0 1 1 3 3 10 10 30 30 100 Total Cumulati ALM Wire Bn o MIO RII RII SOLON Ole R O r lt lt Back Next gt gt Finish Cancel Elms Experiment Creation Wizard Step 9 of 11 _ x Sample Summary Hovering the mouse over a compound in the graph will reveal the complete identity of the compound 4 right click mouse action on the graph or the spreadsheet will offer additional display and export options Export For Recursion Total number of Aligned Compounds 3390 Mass Ys RT Compound Frequency Mass Legend Mass vs RT Color by Frequency rs as 1 7 6 73 Help lt lt Back
28. ew data change the plot view export selected data or export the plot to a e The Summary Report is displayed file as a spreadsheet view when you c Click Next have more than 30 samples Ll workflow Type Analysis Significance Testing and Fold Change Step 1 of 8 xj Steps Summary Report The distribution of normalized intensity values across all samples is displayed in the Profile Plot 1 Summary Report 2 Experiment Grouping MassHunterQual IDENTIFIED_UNIDENTIFIED_COMPOUNDS experiment No of sample s 8 3 Filter Flags 4 Filter By Frequency 5 QC on samples 6 Significance Analysis 7 Fold Change 8 IDBrowser Identifica Log2 Normalized Abundance Values 1 1_pH7_pos_0 1 2_pH7_pos_01 L 1 3_pH7_pos_01 L 1 4_pH7_pos_01 L 3 1_pH7_pos_01 L 3 2_pH7_pos_01 L 3 3_pH7_pos_01 L 3 4_pH7_pos_01 All Samples Select All Rows Help Invert Row Selection lt lt Back Next gt gt Finish Cancel Clear Row Selection Selection Mode Limit To Row Selection Zoom Mode Select Columns Invert Selection Invert Column Selection Clear Selection Clear Column Selection Limit To Selection Reset Filters Reset Zoom Freeze Columns Before Copy view Ctrl C Unfreeze Columns Export Column to Dataset Copy Print Ctrl P Copy view Ctrl C Export As k Print Ctrl P Trellis Publish Catview Export As gt Color By Venn Properties Ctrl R Properties Ctrl R 24 MPP Application Guide Steps 2 Defi
29. ia Demo Malaria Demo samples tar MPP Application Guide 37 6 Perform Advanced Operations 38 The operations available in the Workflow Browser provide the tools necessary for analyzing features from your mass spectrometry data depending upon the need and aim of the analysis the experiment design and the focus of the study This helps you create different interpretations to carry out the analysis based on the different filtering normalization and standard statistical methods MPP Application Guide MPP Application Guide BioCyc Pathway Genome Databases Includes BioCyc Pathway Genome databases from the Bioinformatics Research Group at SRI International used under license http www biocyc org Citation based on use of BioCyc Users who publish research results in scientific journals based on use of data from the EcoCyc Pathway Genome database should cite Keseler et al Nucleic Acids Research 39 D583 90 2011 Users who publish research results in scientific journals based on use of data from most other BioCyc Pathway Genome databases should cite Caspi et al Nucleic Acids Research 40 D742 53 2012 In some cases BioCyc Pathway Genome databases are described by other specific publications that can be found by selecting the database and then going to the Summary Statistics pages under the Tools menu The resulting page sometimes contains a citation for that database 39 www agilent com In
30. if applicable by clicking the Import parameters from samples a4 button File Home Insert Page Formi Data Revier View fe Samples B c Infection Test Gro Not Infected Value 1 Not Infected Value 2 Not Infected Value 3 Not Infected Value 4 Infected Value 1 Infected Value 2 Infected Value 3 Infected Value 4 1 1_pH7_pos_01 1 2_ pH _pos_01 1 3_pH _pos_01 1 4 pH7_pos_01 3 1_pH _pos_01 3 2_pH _pos_01 3 3_pH _pos_01 3 4 _pH7_pos_01 Wl oin mols amp wih hr Oo CEEI Malaria Application Ready 7 Filter the molecular features by abundance mass range number of ions per feature and charge state in the MS Experiment Creation Wizard Step 7 of 11 Elms Experiment Creation Wizard Step 7 of 11 a e x id Y gt Malaria Application tsy Microsoft Excel os Addl 9 o P X D up Test File Edit Format wiew Help Samples Infection Not Not Not Not Infected Infected Infected Infected 3 1_pH7_pos_o1 3 2_pH _pos_oOl 3 3_pH _pos_oOl 3 4_pH _pos_o01 NNN NHN KF FF KF FE a Mark the Minimum absolute abundance check box under Abundance filtering b Type a value of 5000 counts c Clear the Limit to the largest and Minimum relative abundance check boxes x Filtering experiments Number of ions filter is Number of Model ions Filtering during the data import process may be used to reject low intensity data or restrict the range of data After data is imp
31. itability System suitability involves collecting data to provide you with a means to evaluate and compensate for drift and instrumental variations to assure quality results Techniques employed by your Agilent MassHunter software include 1 retention time alignment 2 intensity normalization 8 chromatographic deconvolution and 4 baselining to produce the highest quality results The best results are achieved by maintaining your instrument and using good chromatography Sampling methodology Improved data quality comes from matching the sampling methodology to the experimental design so that replicate data is collected to span the parameter values for each parameter A larger number of samples appropriate to the population under study results in a better answer to your hypothesis An understanding of the methodologies used in sampling and using more than one method of sample collection have a positive impact on the significance of your results Where to find more information to help you prepare for your experiment Step by step detail of the process for preparing for your experiment and performing an unbiased differential analysis is presented in the Metabolomics Discovery Workflow 5990 7067EN MPP Application Guide 5 2 Find the Features in your Data Before you analyze your data with MPP the features compounds in your data must be extracted into compound exchange CEF files The features in your sample data are found and
32. m Fo bio 1A i n M ra A Poo ao wt an a7 z pected aar 1 D D l 5 f 5 Li 11 a iE 16 Lero dalal amp ER ep i Boo 1 40 gt ji EZE IEH z F PEF PET s e i OP Bete at oe 7 TAHA OD UE brivad 44 ey T FET J E i TELE FI a eiL 18 fae rah i ETE T i jjE 1 1365 2 i J e a tJi 7 108 1 aiaia l i LFE Lite lif F 3E RE pac Pd SERRE SE el fetes eee am feed va Piere Tec vie Mita I Foii charge out of H J o mgp ifeced l eem aiae tran coe ka tee eee Deeg ered Ped Charge Cig hs a a Meja Tapia anne Any x z Danei a ber beii nr Chee reda A Pree a aara Ta apiy hh er jea T b g a praka aT lee a ga Pok ee pa l aha Pha an Gapa Yami mil a bah la Hp Pd Ta l Summary Eart He rast bap ro ante paa Fa ihe r fjerm a Eep FPE i an d DD erie aata naa pa oun all GOS 3 ie Flan set bergi i Pile By Pree Ueto eine bes ery APRA 2 OOO oi oe prhe COLT Soypmgmare CE Mii Ted Direi ee Fa Puia Calera Bjal harar i Dirman hifa r aa E iii i at T i Th i a aE a 122 wP bi w E x Comet id praka eaer ia pi Ei wal Las i a oor bs farce 3 1 g P 1 an penn sa TIT s TAE 4E 04 SOE 05 T EIE 0 6 47 0 FERFE Ay tee TSi A 8S6 03 A PIE 01 Femo eet oFo odd 410 O03 TIEG B2iEG LORE F E D LEEGI BTTE 07F0 0947 ml iPS 933 LobE oa isi Fedo d sm 0 i FE o1 AFH ao oat yig hi aby ee 480 00 ME FISE 01 A 18 0 Daor 11 ean Bi ate I ipe 2 I
33. nce Analysis 7 Fold Change Filter Flags If flag values are present entities are Filtered based on their flag values Otherwise entities are filtered based on their signal intensity values To change the filter criteria click on the Re run Filter button 8 IDBrowser Identifica Displaying 2107 out of 3390 entities where atleast 2 out of 8 samples have flags in P M Normalized Intensity Values Infected Not Infected Infection lt lt Back Next gt gt Finish Cancel 26 MPP Application Guide Steps 4 Filter the remaining entities in your samples based on their frequency of occurrence among the samples and conditions Filter by Frequency Step 4 of 8 fritter Parameters x Filtering Conditions Retain entities that appear in at least 100 0 of all samples of samples in only one condition of samples in at least one condition of samples within each condition Cancel Ll workflow Type Analysis Significance Testing and Fold Change Step 4 of 8 a og Detailed Instructions Review the summary plot Click Re run Filter to enter parameters into the Filter Parameters dialog box Type 100 in the Retain entities that appear in at least Click of samples in at least one condition Click OK Review the profile plot You are encouraged to repeat the Re run Filter until you obtain the best results for your experiment
34. ne or adjust the sample grouping with respect to the independent variables and the replicate structure of your experiment Experiment Grouping Step 2 of 8 el workflow Type Analysis Significance Testing and Fold Change Step 2 of 8 Detailed Instructions a Click Add Parameter to define or adjust your experiment grouping Follow the steps in Define the sample grouping with respect to the independent variables and the replicate structure of your experiment in the MS Experiment Creation Wizard Step 6 of 11 on page 14 Click Next when you have completed your experiment grouping Comments Note In order to proceed to the next step at least one parameter with two parameter values must be assigned An independent variable is an essential element constituent attribute or quality in a data set that is deliberately controlled in an experiment An independent variable is referred to as a parameter and is assigned a parameter name Steps Experiment Grouping 1 Summary Report 2 Experiment Grouping 3 Filter Flags Experiment parameters define the grouping or replicate structure of your experiment Enter experiment parameters by clicking on the Add Parameter button You may enter as many parameters as you like but only the first two parameters will be used For analysis in the guided workflow Other parameters can be used in the advanced analysis You can also edit and re order parameters and par
35. old Change Step 7 of 8 type a value to change the Fold change cut off The default value is 2 0 A larger cut off value passes a smaller number of entities through to the final results c Select a value for the Minimum number of pairs of conditions that must have entities with a fold change greater than the cut off The default value is 1 d Click Next l avk per kaise hapai mo r Ipe ared hi bagr er ed BY xj fold Chere wore Pet ei BI TS CCI pE E Rages Pop et i j ph Me ree heed targa cohort nag che Paid hage oo kie et re es aT Depr DE er oe ee et ee ee et Bs ee rete pe sth a Pe eed coor De ___ i ikad BAd aH n ee a a Bpl ITEE i j fag x Lt ia GLH TI LI Pak charge ol a E Es Hra of pee j Cop J d rs awa oo ne depending on your experiment setup it is skipped using the Malaria Demo f your experiment has a parameter that contains at least three parameter values the Fold Change step is available Fold change is a signed value that describes how much an entity changes from its initial to its final value For example when an entity changes from a value of 60 toa value of 15 the fold change is 4 The quantity experienced a four fold decrease Fold change Is the ratio of the final value to the initial value Fold change analysis Is used to identify entities with abundance ratios or for example differences between a treatment and a control that are in excess of
36. orkflow Type Analysis Significance Testing and Fold Change Step 8 of 8 IDBrowser Identification To identify the Entities that passed the fold change cut off with IDBrowser click on the IDBrowser Identification button Steps 1 Summary Report aa ial al Identify Entities with IDBrowser y 3 Filter Flags Compound i a Regulation Fe amp Fe _ log 4 Filter By Frequency C19 H18 N6 010 55 i 1 19E 11 18910 66 5 QC on samples C23 H10 N4 022 i 9 68E 05 3 08 a C14 H6 N6 05 53 2 89E 04 2 16 6 Significance Analysis Ifc34 H8 N2 018 i 4 65E 05 2 60 7 Fold Change C13 H22 012 55 2 01E 04 2 49 791 957 1 0 321875 f 2 98E 05 2 63 C20 H15 CIN4 0165 i 5 66E 14 34984 03 C14 H16 N2 01055 6 38E 13 121746 46 C14 H12 N2 09 3 1 37E 04 2 01 793 9276 0 31875 1 48E 10 41750 41 C12 H8 N4 09 54 5 30E 03 2 18 C3 H6 05 53 f 2 34E 03 2 00 C10 H3 CI 015 4 31E 03 2 93 C16 H CI N4 01154 4 54E 14 101585 82 C6 H8 N2 03 55 1 58E 03 3 16 135 9332 0 330125 1 43E 03 2 21 C12 H5 CI N4 09 4 2 28E 15 43339 32 8 IDBrowser Identifica 974 3772 0 37175 1 19E 09 127095 63 C13 H23 N9 025 85E 07 2 56 C13 H14 N40 8 58E 12 81736 20 C16 H23 N5 010 53 1 75E 08 25534 73 C18 H9 N3 010 2 07E 10 235596 20 C4 H10 025 4 28E 13 232076 61 C19 H13 N110 i 3 53E 10 27617 06 C14 H
37. orted there are several Filtering options that may be applied Filter by Frequency Abundance Variability Flags and Annotation For AMDIS Abundance filtering J Minimum absolute abundance 5000 counts compounds J Minimum relative abundance Yo I Limit to the largest Retention time Filtering JV Use all available data Min ETIO 0719 fo o719 Max RT 15 4101 fis 4101 Number of ions Minimum number of ions 2 Single ion compounds only Charge states Multiple charge states forbidders Mass Filtering I Use all available data Min Mass 50 0167 50 00 Max Mass 2589 16 1000 C All charge states permitted Multiple charge states required Dart maa aa Help lt lt Back Next gt gt Finish Cancel sf Malaria Application tsy Notepad Creating and editing experiment parameter groupings may be more convenient for you using Microsoft Excel Save your file as a tsv file O x Test Group Infected Infected Infected Infected Value Value Value Value value value value value WNP awh The filtering parameters dialog box is unique for each experiment type More information may be found in the online Help MassHunter Qual as the selected data source used in this example presents the most active fields Filtering during the data import process may be used to reject low intensity data or restrict the range of data In a Find by
38. re Ed fs meann eias Interactive views for selections at Chast Preictien a made in the Experiment Navigator eee i a iif VA WN y WAAN VV Color ih related to the current view ped debacle We Information related to the current view and a memory monitor Click the garbage canto reduce memory usage 36 MPP Application Guide 5 Save your project Save your current analysis as a TAR file for archiving restoration of any future analysis to the current results sharing the data witha collaborator or sharing the data with Agilent customer support Steps Detailed Instructions Comments 1 Export your project to a TAR file a Click Project gt Export Project e You have completed creating your b Mark the check box next to the project and analyzing an experiment you wish to save experiment It is recommended to c Click OK archive your progress by exporting xi VORT PEES Select experiments bo export with the project All the contents of the experiment will be exported and can be imported later W Malaria Demo samples Help Cancel d Select or create the file folder e Type the File name f Click Save save hhh Xe Save in rr Malaria Demo d EE El Recent Items File name Malaria Demo samples tar Save Files of type rar archive tar z Cancel g Click OK x ve i Exported project to C Program FilestAgilentiMassHunter Workstation isamples Malar
39. rediction model using imported retention time raining data Sis Select Identified when the Experiment name Malaria dema compounds have been identified by compound formula and or Analysis type Mass Profiler Professional CAS b numper Experiment type Unidentified E e Select Combined Identified workflow type JAnalysis Significance Testing and Fold Change Unidentified when you are unsure if the data has been identified in full or in part or when MassHunter Qualitative Analysis has been previously used to identify some of the compound features Experiment notes Malaria LCMS ESI pH 7 Table1 Table of selections and entries for the New Experiment dialog box Dialog Box Feld Your Choices Comments Experiment name Edit field to describe this experiment Analysis type Mass Profiler Professional Mass Profiler Professional must be selected lt other choices depending on Order IDs gt Experiment type Combined Identified and Unidentified lt see next table gt Identified Unidentified Analysis Significance Testing and Fold Change Glass Prediction Build and Test Model Data Import Wizard Experiment notes PO Edit field to enter other experimental notes MPP Application Guide 9 Steps Detailed Instructions Comments Table 2 Table of data sources and file extensions based on Experiment Type Experiment Type Data Source Fe types o UUU Identified MH Quant Compounds identified by MassHunter Quan
40. reparation includes grouping filtering alignment normalization and baselining Flow Chart of the MS Experiment Creation Wizard r b E F TE E E EE F TF Select Data Source Grouping Sample Summary Step 2 Select Data to Import Step 10 Step T Normalization Fastest Criteria Steps 3 amp 4 shopped Go to Stepi Step 8 Step 11 Alignment Baselining Options fl Go to Step9 Create an initial analysis Figure 3 MS Experiment Creation Wizard Steps Detailed Instructions Comments 1 Select the data source that a Click MassHunter Qual and select e Ifyou are using your own data set generated the molecular features Homo sapiens for the Organism if you click the source of your sample for your experiment in the MS are using the Malaria Demo data set files and select the Organism of Experiment Creation Wizard Step b Click Next the sample files or select None 1 of 11 e Note that selecting an Organism is most important when you use the Pathway Analysis features of MPP Elms Experiment Creation Wizard Step 1 of 11 xj Select Data Source Choose the data sources that will be used For the experiment MassHunter Qual MassHunter ICP MS C AMDIS Generic Organism Homo sapiens i Finish Cancel MPP Application Guide 11 Steps Detailed Instructions Comments 2 Select the molecular feature a Click Select Data Files e The file
41. sed to import organize and analyze the data you acquired Your unbiased differential analysis experiment may include the following steps with MPP beginning at step four 1 prepare for your experiment 2 acquire your data 8 find the spectral features 4 import and organize your data 5 create your initial analysis 6 identify the features 7 save your project and 8 perform advanced analysis operations Figure 1 on page 2 shows the Agilent tools in your experiment Workflow for Unbiased Differential Analysis Prepare for an Import and Organize Data Experiment Create an Initial Analysis Identify Features Experiment Design Find Features Advanced Operations as Compounds amp Data Acquisition Spectral Features Statistical Data Analysis Identification Experiment Design Statistical Analyses Hypothesis Interpretations Natural Variability Pathways Replicate Sampling Filtering System Suitability Class Prediction Sampling Methodology O Additional features GC MS Qu A Mass Profiler MassHunter LC MS p T as Professional 1D Browser with DA Reprocessor Figure 1 The steps involved in an unbiased differential analysis How do use MPP to analyze my data MPP helps you analyze your data through the use of sequential dialog boxes and wizards as shown in Figure 2 MPP Application Guide project and an experiment Identify features as compounds Order and Save your group the project and data files experiment s Fil
42. specified cut off or threshold value Fold change is calculated between the conditions where Condition 1 and another condition Condition 2 are treated as a single group 30 MPP Application Guide Steps Detailed Instructions 8 Export the significant entities in a Review the summary plot your experiment for identification b Highly recommended Click Back to ID Browser Identification Step 8 make adjustments to prior steps in the of 8 workflow to improve the results c Click IDBrowser Identification to export your entity list to Agilent MassHunter ID Browser ID Browser is started and automatically prompts you to set up your identification method Comments Processing your entities with ID Browser performs the following automatically save the selected entity list into a CEF file format open Agilent MassHunter ID Browser and import the saved CEF file for identification Once identification is completed ID parameters Browser returns an identified CEF wees otras ene TT 5 file This CEF file is imported into o bromera tp he a ahem i iene at the MPP experiment and imane a es a j ST E annotations are automatically er pomene En F i updated pinne ne z r E Fe Ta i i TE a E Se Te Ee fT ST d Select the compounds to identify and mark the identification method for your experiment in the Compound Identification Wizard dialog box e Click Next 8 compound Identification Wizard E
43. ss MPP Application Guide 33 Steps Detailed Instructions Comments h Review and make adjustments to the entity identifications as necessary using the ID Browser interface Click Save and Return Saveand Return to export your entity list back to your experiment in MPP You are automatically returned to the MPP user interface Wlagilent MassHunter ID Browser B 05 00 i Oo x File Edit view Identification Method Configuration Help A Run ID Wizard ale He amp abe Ge Save and Return il MS Spectrum Resuks x i MS Peaks One MFE Spectrum 0 316 min x amp Structure Viewer x ze t QR mz Abund Abund Nom z Sat Eod cc 103 Cpd 1 C19H18 NG 010 55 0 316 MFE Spectrum 0 316 min T E E et 650 9758 ssd wae T arse rt 45 4 3 5 3 25 2 1 5 1317 9713 1 2M NH4 700 800 900 1000 1100 1200 1300 Counts vs Mass to Charge m z LMS Spectum Resuts f amp Compound List x if Cpd Label Y Name M Formula Td Score Mass Y AvgMass Y Std Dev Y Mass DB 2 aP a e E e e a eeen m e a 34 MPP Application Guide Steps Detailed Instructions Comments j Review your identified entity list in the ID Browser Identification results The molecular formula now replaces the mass and retention time for identified entities in the compound column k Click Finish when you have completed the ID Browser Identification fl w
44. t 2 Z Axis Component 3 Color by Infection Infected Not Infected Description Legend 3D PCA Scores Algorithm Principal Components Analysis Parameters Column indices 1 8 Pruning option numPrincipal Components 4 Mean centered true Scale true 3 D scores true PCA on Columns lt lt Back Next gt gt Finish Cancel 28 MPP Application Guide Steps Detailed Instructions Comments 6 Assess the differential significance a Review the summary plot e of your samples Significance b Highly recommended Click Back to Analysis Step 6 of 8 make adjustments to prior steps in the workflow to improve the results c Customize the window panes d Move the p value cut off slider s or type a value to change the p value cut off value s A larger p value passes a larger number of entities Elie e ip tee eee Deeg ard Ped harge Clg ha a Jeja Tapia anne Any x z A ie B ri en prebi d ee a i Ta jay a aer jeah a T h a a rka aT ie ie fga Pab Aem p ih mah Pi eor fapa Yona mil aid bab abha Hi feii nr L Bumar Beart te rast bap ro antie paa Fe the P fpi Ga Camisa B ad a GiT eis aig n pia io LA 1 fie Flan k Jeet Deecrgimorn ie By heer Seid ir te LO mg pee ENT merges CE Mape Trier Lorre Hor Bamps sey P Pa urga yal hamar TE a ras ran an r ra a oF i a i ft a Feri mI is Ti K 7 re gt if ih i tj FY ii
45. tabase eee amp amp amp amp amp amp Generate Formulas xj aa Charge states if not known gt Agprepetes I Dimes eg 2M H Charge stat fi EEEa l Trimers e g 3M H Maximum number of peaks to search when peaks are not specified graphically SSS E x Ee townie Aggregates l Dimes e g 2M H Charge stat fi i I Times eg 3M H I A C Scoing SeachMode T Contribution to overall score Mass score Isotope abundance score Isotope spacing score 50 00 Retention time score Expected data variation 20 mDa MS isotope abundance MS MS mass 5 0 mDa Retention time MS mass Negative lons m lon search mode Which database entries should be examined when searching masses from simple ions C Neutral entries Cation or anion entries This choice is not applicable to CSV databases Negative lons i Search Results IV Limit to the best 10 hits 32 MPP Application Guide Steps Detailed Instructions Comments El compound Identification Wizard g Click Finish when you have the method set up for your experiment ID Browser automatically begins identifying your entities and shows a progress bar Operation in Progre
46. ter align and normalize Export your the sample project dala Figure 2 Overview of the wizards that help you use MPP Where do get more information MPP Application Guide The Agilent Metabolomics Workflow Discovery Workflow Guide and the Agilent MassHunter Mass Profiler Professional User Manual see below provide you with additional detail techniques and explanations to perform advanced analysis operations e Agilent MassHunter Mass Profiler Professional User Manual Agilent publication January 2012 You can find a PDF copy of the user manual in the MPP installation folder C Program Files Agilent MassHunter Workstation Mass Profiler Professional docs manual e Agilent Metabolomics Workflow Discovery Workflow Guide Agilent publication 5990 7067EN Revision B October 2012 e Agilent Metabolomics Workflow Discovery Workflow Overview Agilent publication 5990 7068EN Revision B October 2012 1 Prepare for your Experiment An experiment consists of the analysis of a set of replicate samples collected over a range of well defined parameters treatments and or exposures known as independent variables including parameter controls representing minimal or normal perturbations control samples The results of changes observed in the samples is designed to provide an answer to your hypothesis The hypothesis may be proved or disproved by analyzing the correlation of the independent variables on the resulting expression
47. the quality of an entity within a sample A flag indicates if the entity was detected in each sample as follows Present means the entity was detected Absent means the entity was not detected and Marginal means the signal for the entity was saturated This filter removes irreproducible entities from further consideration by your analysis MPP Application Guide 25 Steps Detailed Instructions encouraged to repeat the Re run Filter until you obtain the best results for your experiment Click Next Comments d Clear the Absent check box This flag The number of entities displayed is useful when you want to identify above the profile plot is expected to missing entities in the sample data decrease as you progress through e Clickatleast__ out of X samples the workflow have acceptable values The X is replaced in your display with the total A one hit wonder is an entity that number of samples in your data set appears in only one sample is f Type 2 in the entry box By setting this absent from the replicate samples parameter to a value of two or more and does not provide any utility for one hit wonders are filtered statistical analysis g Click OK h Review the profile plot You are Ll workflow Type Analysis Significance Testing and Fold Change Step 3 of 8 Steps 1 Summary Report 2 Experiment Grouping 3 Filter Flags 4 Filter By Frequency 5 QC on samples 6 Significa
48. this book The Agilent G3S835AA MassHunter Mass Profiler Professional Software Application Guide presents additional detail of the software interface and helps you use MPP with your data Agilent Technologies Inc 2012 Revision A November 2012 G3835 90011 ofthe Agilent Technologies
49. titative Analysis Chemstation FIN Compounds identified by Chemstation Quantification or Screener processes Generic 7 Entries identified by Compound column C Formula column D CASID column E Unidentified MH Qual Find By Molecular Feature Extractor MFE MH Qual GC Scan Find by Chromatographic Deconvolution ICP MS Identified by ICP MS software AMDIS ELU Components identified by AMDIS that are not identified by an AMDIS target librar Generic Entries NOT identified by Compound column C Formula column D CASID column E TXT Combined MH Qual Find By Molecular Feature Extractor MFE and Find By Formula MH Qual GC Scan Find by Chromatographic Deconvolution and Library Search ICP MS Identified by ICP MS software AMDIS Targets and components discovered by AMDIS ELU Generic Combination of entries identified by and not identified by Compound column C Formula column D CASID column E e f you selected Analysis Significance Testing and Fold Change or Data Import Wizard for the Workflow type in the New Experiment dialog box you immediately begin the data import process 10 MPP Application Guide Import and organize your data After you set up your project and create an experiment the MS Experiment Creation Wizard Figure 3 immediately guides you through the necessary steps to organize your experiment import your data define your experiment variables and prepare your data for analysis data p
50. ur parameter values Elms Experiment Creation Wizard Step 6 of 11 Detailed Instructions a Click any one value under the parameter column to select the whole parameter column b Re order the parameter column click the Left Em or Right m button Experiment Grouping Experiment parameters define the grouping or replicate structure of your experiment Enter experiment parameters by clicking on the Add Parameter button You may enter as many parameters as you like but only the first two parameters will be used For analysis in the guided workflow Other parameters can be used in the advanced analysis You can also edit and re order parameters and parameter values here Displaying 8 sample s with 3 experiment parameter s To change use the button controls below Se joe H _pos_O1 Not Infected pH _pos_01 Infected H _pos_O1 Not Infected H _pos_01 Infected H _pos_01 Not Infected pH _pos_ ol Infected H7_pos_01 Not Infected H7_pos_01 Infected Elorder Parameter Yalues x Order Parameter Yalues The order in which the conditions appear in the window below will be the order in which they are displayed in views For example in a Profile Plot the first listed condition in the window below will be the first condition displayed on the X axis To re order the conditions select the condition and move it up or down by clicking on the
51. value for the selected samples Not Infected cei Add Edit Experiment Parameter x Grouping of Samples Samples with the same parameter values are treated as replicate samples To assign replicate samples their parameter values select the samples and dick on the Assign Values button and enter the value for the group Set the parameter type to numeric to interpret the parameter values as numbers Parameter name infection Parameter type Non Numeric bd Ses O Parameter Values Not Infected Not Infected Not Infected Not Infected 3 1_pH7_pos_01 3 2_pH7_pos_01 3 3_pH7_pos_01 xi Enter a value for the selected samples finfected tt s lt s a Detailed Instructions f Type the value for your first grouping in the Assign Value dialog box For the Malaria Demo type Not Infected Click OK Click your replicate Samples that share the same second parameter value in your data For example e 3 1 _pH _ pos 01 e 3 2 pH _pos 01 e 3 3 pH pos 01 e 3 4 pH pos 01 Click Assign Value Type the value for your second grouping in the Assign Value dialog box For the Malaria data type Infected Click OK Repeat the value assignment steps with your own data until you have assigned a parameter name type and value to all of your samples Review your entries and grouping assignment accuracy in the Add Edit Experiment Parameter dialog box Repeat the value assignm

Mass Profiler Pro Application_Guide F8.book

Contents

Download Pdf Manuals

Related Search

Related Contents