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Mass Profiler Pro Application_Guide.book

1. MS Experiment Creation Wizard Step 9 of 11 Total Samples 8 1400 1200 wo E 5 1000 o a E s800 5 e 600 zZ 400 0 1 8 7 6 5 4 3 2 1 Frequency onset tm t0 30 30 10 Cumulative Total 8 539 472 51 10 _ i 2 4312 7 25 237 1 2 1 0 ee 6111 6 225 210 7 6 1 1 1350 7461 5 220 208 6 4 1 1 1100 8561 4 283 277 2 2 2 o 1132 9693 3 372 361 5 3 1 2 1116 10809 2 590 569 10 8 2 i 1180 11989 1 1514 1472 30 8 4 o 1514 13503 z Sample Summary From the Entities tab us options to manually merge entities Spectra of selected entities Compour aa Frequency tb d a the frequency of aligned compounds across all the samples Mass vs tab display has the summary of aligned compounds present or absent in individual samples re displayed to help merging Total number of Aligned Compounds 4000 Entities Compound Frequency Mass vs RT Mass Profiler Professional Application Guide en a re a Click the Compound Frequency tab b 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 sa
2. 2_pH _pos_O1 1 2 3 4_pH _pos_01 I 2 3 4 3_pH _pos_O1 p p p _pH _pos_O1 p p p 4_pH _pos_O1 Add Parameter Edit Parameter Delete Parameter lt lt Back Next gt gt Finish Cancel SEZ Experiment Parameter i 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 click 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 ad Samples Parameter Values os 0 Assign Value Clear Cancel 18 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 1 1_pH7_pos_01 1 2_pH _pos_01 1 3_pH _pos_01 1 4 _pH _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 qualit
3. make adjustments to prior steps in the workflow to improve the results c Click Next Fl 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 H _pos_01 Displaying 8 out of 8 samples retained in the analysis Not Infected 2_pH _pos_01 Not Infected 3_pH _pos_O1 Not Infected H7_pos_01 Not Infected Infected Infected 8 IDBrowser Identifica Infected Infected Component 1 Axis Component 2 Color by Infection Infected E Not Infected Description Legend 3D PCA Scores Algorithm Principal Components Analysis Parameters Column indices 1 8 Pruning option numPrincipal Components 4 Scale true 3 D scores true PCA on Columns Z Axis Component
4. C20 H15 CI N4 76 85 633 9889 8 Cpd 8 C14 H16 N2 010 55 C14 H16 N2 01 79 72 531 9396 E 9 Cpd 9 C14 H12 N2 09 53 0 C14 H12 N2 09 77 01 447 9698 a 10 Cpd 10 0 319 793 9276 aft gt k 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 Click Finish when you have completed the ID Browser Identification The Analysis Significance Testing and Fold Change workflow is now complete and you are immediately returned to the main MPP interface 40 Mass Profiler Professional Application Guide 4 Create your Initial Analysis Steps Detailed Instructions Ll workflow Type Analysis Significance Testing and Fold Change Step 8 of 8 Steps IDBrowser Identification To identify the Entities that passed the fold change cut off with IDBrowser click on the IDBrowser Identification button 1 Summary Report Comments 2 Experiment Grouping Identify Entities with IDBrowser ibrowse identification 3 Filter Flags 4 Filter By Frequency C19 H18 N6 01055 4 02E 10 1 19E 11 9 68E 05 down 18 0 18910 66 5 QC on samples C23 H10 N4 022 1 29E 03 a C14 HG N6 05 3 3 48E 03 2 89E 04 ra 05 3 08 1 62 i 2 16 2 16 111 6 Significance Analysis cog H8 N2 018 6 28E 04 4 65E 05 ul a260 2 60 1 38 2 01E 04 7 Fold Change C13 H22 012 5 2 52E 03 F
5. 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 lt lt Back Next gt gt Finish Cancel Elms Experiment Creation Wizard Step 10 of 11 i 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 H H H H H H H lt lt Back Next gt gt Finish Cancel 26 Mass Profiler Professional Application Guide Steps Detailed Instructions 11 Compare the features in each a Click the Baseline to _ of alll sample to the response of each samples button feature across multiple samples or b Select median for the Baseline to the control samples in the MS ___ of all samples Experiment Creation Wizard Step c Click the Finish button lt lt 5e 11 of 11 Elms Experiment Creation Wizard Step 11 of 11 4 xj Baselining Options There are four baseline options 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 Baseline each entity to median mean across samples or control samples These options
6. value For the group Set the parameter type to numeric to interpret the parameter values as numbers Parameter name infection Parameter type Non Numeric v Parameter Values 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_01 Infected 3 3_pH7_pos_01 Infected 3 4_pH 7_pos_01 Infected alear Assion Value Cancel Detailed Instructions p Repeat Add Parameter if your data has more than one independent variable Click Add Parameter Repeat the steps above until you have assigned a parameter name type and value to all of your data Review step 5 OPTIONAL Re order your parameter values and step 6 OPTIONAL Saving and importing experiment grouping information in a spreadsheet These steps provide advanced instructions to manage your parameters and parameter name assignments using the wizard toolbar and a spreadsheet application Click Next when you have completed your experiment grouping Comments You may change the value of any sample or group of samples highlight the sample and click Assign Value or Clear Note You may add grouping or change your grouping during the Analysis Significance Testing and Fold Change Wizard and at any time thereafter Elms Experiment Creation Wizard Step 6 of 11 x Experiment Grouping Experiment parameters define the grouping or r
7. 0 02 90 P lt 0 01 P lt 0 0050 P lt 0 0010 82 78 68 FC gt 1 1 783 105 90 82 78 68 T i g D 2 S E 1 4 03E 10 18910 66 a 693 9807 0 32 9 68E 05 1 29E 03 3 08 a 433 9571 0 3 18625 2 89E 04 3 48E 03 2 16 611 9776 0 32137498 4 65E 05 6 28E 04 2 60 529 9748 0 32137498 2 01E 04 2 52E 03 2 49 791 9571 0 321875 2 98E 05 4 07E 04 2 63 633 9917 0 31725 5 66E 14 1 30E 11 34984 03 531 9423 0 31775 6 38E 13 5 31E 11 12 1746 46 447 972 0 322 1 37E 04 1 79E 03 2 01 793 9276 0 31875 1 48E 10 3 26E 09 41750 41 479 9197 0 32762498 5 30E 03 4 63E 02 2 18 217 9361 0 328625 2 34E 03 2 31E 02 2 00 397 9163 0 32937503 4 3 1E 03 3 91E 02 2 93 593 8678 0 32750002 4546 14 _1 30E 11 101585 82 315 9133 0 330125 1 58E 03 1 61E 02 3 16 135 9332 0 330125 1 43E 03 1 49E 02 2 21 511 8639 0 33075 2 28E 15 2 10E 12 43339 32 974 3772 0 37175 1 19E 09 1 94E 08 127095 63 369 1682 0 3785 7 85E 07 1 11E 05 2 56 biatah 242 1197 0 3815 8 58E 12 3 33E 10 81736 20 2 a 541 0607 0 419 1 75E 08 2 58E 07 25534 73 Select pair Not Infected Vs infected A497 NIAAA AL17AQGQQ AFFIN d 12F_NG 25590R zni p value cut off es Fold change cut off HK 2 0 Klworkflow Type Analysis Significance Testi
8. 3 16 3 16 1 66 135 9332 0 330125 1 49E 02 1 43E 03 down 2 21 2 2 1 1 14 511 8639 0 33075 2 10E 12 2 28E 15 down 16 00 43339 32 15 40 974 3772 0 37175 1 94E 08 1 19E 09 down 16 00 127095 63 16 96 369 1682 0 3785 1 11E 05 7 85E 07 up 2 56 2 56 1 36 242 1197 0 3815 3 33E 10 8 58E 12 down 16 00 81736 20 16 32 541 0607 0 419 2 58E 07 1 75E 08 down 16 00 25534 73 14 64 427 0293 0 45 174998 4 13E 09 2 07E 10 down 16 00 235596 20 17 85 122 0412 0 46225 4 60E 11 4 28E 13 down 16 00 232076 61 17 82 411 1307 0 55525 6 22E 09 3 53E 10 down 16 00 27617 06 14 75 343 0845 0 58125 7 44E 10 2 43E 11 down 16 00 218809 95 17 74 274 5602 0 62724996 2 25E 02 2 26E 03 up Cara reed 1 18 623 0012 0 6505 1 02E 09 3 56E 11 down 16 00 49757 84 15 60 107 0375 0 71250004 3 29E 10 7 90E 12 down 16 00 214511 75 17 71 545 9329 0 73424995 1 72E 09 6 76E 11 down 16 00 42292 68 15 37 850 7883 0 74450004 1 76E 11 9 57E 14 down 16 00 291330 66 18 15 265 9593 0 74725 2 36E 10 5 26E 12 down 16 00 3825816 50 21 87 584 8299 0 75325 2 36E 10 5 41E 12 down 16 00 307682 91 18 23 559 0709 0 929 9 78E 10 3 31E 11 down 16 00 49340 76 15 59 427 0292 0 936 _1 02E 09 _ 3 67E 11 down _ 16 00 __ 490408 84 18 90 663 1087 1 03475 4 72E 09 2 52E 10 down 16 00 315417 03 18 27 122 048 1 066625 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 54384 98 15 73 221 0385 5 0220003 9 15E 1
9. 8 IDBrowser Identifica Normalized Intensity Values Infected Displaying 2107 out of 3390 entities where atleast 2 out of 8 samples have flags in P M Infection Not Infected Help lt lt Back Next gt gt Einish Cancel Mass Profiler Professional Application Guide 31 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 Filtering Conditions of all samples lriter Parameters ue Retain entities that appear in at least 100 0 of samples in only one condition of samples in at least one condition of samples within each condition el workflow Type Analysis Significance Testing and Fold Change Step 4 of 8 Cancel Detailed Instructions oa 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 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
10. List of operations relevant for the Frequency 12 cenam Project Navigator Tii ee ce a i 3 on zat i thi experiment organized into sequential 8 3 1 Control 250 ISFOL MEAE iment lel groups of operations for the analysis J iele the current project 3 4_Control_250 Analysis a 1_Infected r ae ae Statistical Analysis 2 3_Infected_000 10 Filter on Volcano Plot 2 4_Infected_o00 Fold Change O 4 1_Infected_2s0 Clustering 2_InFected H ane a Find Similar Entities O 4 4_Infected_2so wu Filter On Parameters H E Interpretations 5 Principal Component Analysis ii All Samples S Find Minimal Entities QI Infection Treatment Non averaged s Qi Infection Treatment 5 aS z Qui Infection Treatment S tates made in the Experiment Navigator saa aaa E F Filtered on accCalls P fil N A en ls and the Workflow Browser Run Prediction from fle E F Filtered by Fret a Export Prediction Model eGov Experiment Navigator gt Filter By Freq i 0 3 Results Interpretations a eO zwa nova d Lists samples interpretations Find Similar Entity Lists T Test unpair x KENA Export for Recursion a analyses and favorites within 1DBrowser Identification My Favorites a Export for Identification the selected experiment sie SURA Import Annotations Pathway Analysis a Single Experiment Analysis Multi Omic Analysis Launch IPA as Export to Metac Color key related to ee ieia ie the current view Status Bar TE x Information related to
11. and Fold Change Wizard Figure 5 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 Skipped when using the Malaria Demo data set Step 7 Experiment Quality Control on ID Browser Grouping Samples Identification Significance Save your project and Analysis perform advanced tasks Goto Step8 Figure 5 Analysis Significance Testing and Fold Change Wizard Mass Profiler Professional Application Guide 4 Create your Initial Analysis Steps 1 Review the summary of your new experiment Summary Report Step 1 of 8 Detailed Instructions Comments a Review the Summary Report e b Click and right click features on the plot or spreadsheet to review the Familiarize yourself with the tools available to you in the summary report view data change the plot view export e The Summary Report is displayed selected data or export the plot to a as a spreadsheet view when you file have more than 30 samples c Click Next el workflow Type Analysis Significance Testing and Fold Change Step 1 of 8 xj Steps 1 Summary Report 2 Experimen
12. based on Experiment Type Experiment Data Source File Types Comments Type Identified MH Quant Compounds identified by MassHunter Quantitative Analysis ChemStation FIN Compounds identified by ChemStation Quantification or Screener processes Profinder MH Qual CEF Find by Formula MH Qual GC Scan CEF Identify by Unit Mass Library ICP MS CSV Identified by IP MS software AMDIS FIN Compound identified by an AMDIS target library Generic XLS Entries identified by Compound column C Formula column D XLSX CASID column E CSV TXT 12 Mass Profiler Professional Application Guide Table 2 Table of data sources and file extensions based on Experiment Type continued Experiment Data Source Type Unidentified Profinder MH Qual MH Qual GC Scan ICP MS AMDIS Generic Combined Profinder MH Qual MH Qual GC Scan ICP MS AMDIS Generic File Types CEF CEF CEF ELU XLS XLSX CSV TXT CEF CEF CEF ELU XLS XLSX CSV TXT Mass Profiler Professional Application Guide Comments Find by Molecular Feature Extractor MFE Find by Chromatographic Deconvolution Identified by IP MS software Components identified by AMDIS that are not identified by an AMDIS target library Entries not identified by Compound column C Formula column D CASID column E Find by Molecular Feature Extractor MFE and Find by Formula Find by Chromatographic Deconvolution and Library Search Identified by IP MS software T
13. d E Recent Items File name Malaria Demo samples tar Save Files of type Tar archive tar v Cancel g Click OK Mass Profiler Professional Application Guide 43 44 Steps Detailed Instructions Comments Information ia ii he Ai x Exported project to C Program FilestAgilentiMassHunter workstation isamples Malaria Demo Malaria Demo samples tar Mass Profiler Professional Application Guide 6 Perform Advanced Operations 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 Mass Profiler Professional Application Guide 45 www agilent com In this book The Agilent G3S35AA MassHunter Mass Profiler Professional Software Application Guide presents additional detail of the software interface and helps you use MPP with your data This guide applies to MassHunter Mass Profiler Professional Software 13 0 and higher until superseded Agilent Technologies Inc 2014 Revision A September 2014 G3835 90021 fhe Agilent Technologies
14. dialog box Experiment type see also Table 2 determines how Mass Profiler Professional manages the data Select Unidentified when the compounds have only been identified by their molecular features of neutral mass and retention time Select Identified when the compounds have been identified by compound formula and or CAS number Select Combined Identified 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 e If 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 Mass Profiler Professional Application Guide 11 Table 1 Table of selections and entries for the New Experiment dialog box Dialog Box Option Your Choices Comments Experiment name lt none gt 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 Table 2 on page 12 gt Identified Unidentified Workflow type Analysis Significance Testing and Fold Change Class Prediction Build and Test Model Data Import Wizard Experiment notes Enter other experimental notes Table 2 Table of data sources and file extensions
15. may be automatically skipped depending on your experiment setup it is skipped using the Malaria Demo If 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 to a 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 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 Mass Profiler Professional Application Guide 35 Steps 8 Export the significant entities in your experiment for identification ID Browser Identification Step 8 of 8 Steps 1 Summary Report 2 Experiment Grouping 3 Filter Flags 4 Filter By Frequency 5 QC on samples 6 Significance Analysis 7 Fold Change Identifica workflow Type Analysis Significance Testing and Fold Change Step 8 of 8 IDBrowser Identification Detailed Instructions Review the summary plot Highly recommended Click Back to make adjustments t
16. the accuracy of the sample set in describing the characteristics of the population 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 4 Mass Profiler Professional Application Guide System suitability 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 3 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 t
17. used 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 3 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 MassHunter a Interpretations Natural Variability Qualitative Analysis Pathways Replicate Sampling with DA Reprocessor Recursion Filtering System Suitability MFE Class Prediction Sampling Methodology Additional features or GC MS Data MassHunter CEF Mass Profiler MassHunter LC MS Professional ID Browser 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 Mass Profiler Professional Application Guide Import and j A Create an initial Advanced _ or
18. 1 6 42E 01 2 34E 04 21 24594 129 0788 4 16E 01 6 11E 01 6 59E 03 6 21E 01 7 68E 01 3 69E 02 0 046626 410 0033 5 17E 01 8 21E 03 1 09E 03 7 16E 01 1 33E 01 8 72E 03 0 034395 375 9233 3 02E 04 1 58E 02 7 94E 03 6 97E 03 1 72E 01 4 23E 02 403 0491 958 9567 5 67E 01 6 4BE 02 5 98E 03 7 35E 01 4 16E 01 3 43E 02 11 8904 785 9379 1 15E 01 2 36E 04 1 15E 01 3 28E 01 8 62E 03 3 02E 01 53 0632 356 0341 3 03E 03 2 38E 01 3 79E 02 4 94E 02 6 22E 01 1 44E 01 0 7387 633 9917 2 88E 01 2 88E 01 1 23E 05 5 57E 01 6 25E 01 2 11E 04 16 6169 515 9601 1 40E 02 8 94E 03 8 39E 04 9 92E 02 1 35E 01 6 88E 03 157 24 679 967 6 64E 02 1 07E 04 1 77E 01 2 51E 01 4 23E 03 3 76E 01 75 3860 857 9856 4 93E 14 1496 13 8 03E 15 2 23E 12 7 02E 12 2 39E 13 256 327 536 0132 9 59E 01 9 42E 01 1 17E 03 1 00E00 9 99E 01 9 02E 03 0 074912 433 9582 1 09E 17 4 38E 18 2 26E 17 6 99E 16 3 34E 16 8 61E 16 260 136 531 9461 3 62E 01 3 15E 01 2 02E 05 5 76E 01 6 25E 01 2 71E 04 17 2278 693 9808 2 57E 03 6 87E 01 3 75E 07 4 34E 02 8 17E 01 9 15E 06 1 409741 613 9494 8 30E 02 4 70E 01 8 51E 03 2 81E 01 7 27E 01 4 4BE 02 146 798 611 9782 9 03E 03 4 54E 01 5 15E 07 8 31E 02 7 14E 01 1 23E 05 0 66929 529 9749 2 57E 02 2 83E 01 1 01E 06 1 42E 01 6 25E 01 2 33E 05 0 430077 873 96 1 13E 02 4 37E 03 5 01E 03 8 98E 02 1 01E 01 3 20E 02 130 101 791 95
19. 1 1 40E 12 down 16 00 33049 86 15 01 246 0781 5 4035 2 71E 09 1 15E 10 down 16 00 128892 02 16 98 TAA Nae ae ETE TEn Aa EEE PEE TE AA 43207 aal Te aali lt lt Back Next gt gt Einish Cancel 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 Browser returns an identified CEF file This CEF file is imported into the MPP experiment and annotations are automatically updated 36 Mass Profiler Professional Application Guide Steps Detailed Instructions Comments d Select the compounds to identify and mark the identification method for your experiment in the Compound Identification Wizard dialog box e Click Next xl 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 M Database search CSV PCD METLIN J Spectral library search M Molecular formula generator MFG Generate formulas for all compounds Generate formulas only for unidentified compounds Help Next gt gt Finish _ Cancel f Se
20. 2 down 16 00 721451175 C16 H6 N2 0185 1 72E 09 850 7883 0 74450004 1 76E 11 C5 H6 N4 03 3 2 36E 10 9 5 7E 14 5 26E 12 5 41E 12 down 16 z 42292 68 15 oF down 16 se 291330 66 18 gt down 16 00 3825816 50 Cl H9 Cl2 N O8 55 2 36E 10 C23 H17 N3 014 9 78E 10 C18 H9 N3 010 0 936 1 02E 093 3 31E 11 3 67E 11 2 52E 10 down 16 T 307682 91 18 a down 16 00 49340 76 15 59 down 16 00 490408 84 18 90 C28 H25 N 018 4 72E 09 c6 H6 N2 O 5 41E 05 156 5247 2 66825 2 18E 10 3 90E 06 4 29E 12 1 40E 12 down 16 00 315417 03 18 27 down 438 4 30 2 11 down 16 00 54384 98 SENE C5 H11 N5 052 9 15E 11 C8 H14 N4 035 2 71E 09 1 15E 10 down 16 0 33049 86 16 00 128892 02 Mass Profiler Professional Application Guide 41 4 Create your Initial Analysis Steps Detailed Instructions Comments ElMass Profiler Professional My Two Yariable Experiment a BE Jaj xj Project Search View Tools Annotations Windows Help fe 0 0 oaa Project Navigator My Two Yariable Experiment oy Two Variable Data Set Experiments Experiment Setup 2 ii My Two Variable Experiment Quick Start Guide M en u Bar Experiment Grouping e Interpretation Workflow Browser antrai a ontrol on Samples 1 1 control_o00 7
21. 3 Mean centered true lt lt Back Next gt gt Finish Cancel Mass Profiler Professional Application Guide 33 Steps 6 Assess the differential significance of your samples Significance Analysis Step 6 of 8 Workflow Type Analysis Significance Testing and Fold Change Step 6 of 8 Detailed Instructions Review the summary plot Highly recommended Click Back to make adjustments to prior steps in the workflow to improve the results Customize the window panes 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 Steps 1 Summary Report 2 Experiment Grouping 3 Filter Flags 4 Filter By Frequency 5 QC on samples 6 Significance Analysis 7 Fold Change 8 IDBrowser Identifica Significance Analysis Entities are filtered based on their p values calculated from statistical analysis To apply the new p value cut off drag the p value cut off slider or input the new cut off value in the text box You will not be able to proceed to the next step if no entities pass the filter Displaying 81 out of 917 entities satisfying corrected p value cut off 0 05 Test Description Selected Test p value computation Multiple Testing Correction T Test unpaired Asymptotic Benjamini Hochberg y Result Summary pal P lt 0 05 P lt
22. 3 791 9571 0 321875 4 07E 04 C20 H15 CI N4 0165 1 30E 11 2 98E 05 5 66E 14 6 38E 13 up 2 49 2 49 1 82 up 2 63 2 63 1 39 down 16 00 34984 03 15 09 C14 H16 N2 01055 5 31E 11 C14 H12 N2 09 3 1 79E 03 793 9276 0 31875 3 26E 09 1 37E 04 1 48E 10 5 30E 03 down 16 00 121746 46 16 89 up ae 2 01 aoe down 16 00 41750 41 C12 H8 N4 09 54 4 63E 02 C3 H6 05 3 2 31E 02 C10 H3 ClO15 3 91E 02 2 34E 03 4 31E 03 4 54E 14 down 2 is 2 18 A T down 2 00 2 00 1 aa SEE ee down TEE C16 H7 CI N4 01154 1 30E 11 C6 H8 N2 03 55 1 61E 02 135 9332 0 330125 1 49E 02 1 58E 03 1 43E 03 2 28E 15 2 9 down 16 00 101585 82 16 down 3 3 5 1 s down C12 H5 CI N4 09 54 2 10E 12 974 3772 0 37175 1 94E 08 C13 H23 N9 02 5 1 11E 05 i ____te 19E 09 B5E 07 sg 8 58E 12 down 16 a 43339 15 re ars A 00 127095 63 16 26 2 2 ae C13 H14 N40 3 33E 10 C16 H23 N5 010 3 2 58E 07 C18 H9 N3 010 4 13E 09 1 75E 08 2 07E 10 4 28E 13 rE 16 00 81736 20 16 F down 16 co 25534 73 14 24 down 16 00 235596 20 C4 H10 025 4 60E 11 C19 H13 N11 0 6 22E 09 C14 H13 N7 025 7 44E 10 3 53E 10 2 43E 11 2 26E 03 down 16 0 232076 61 E down 16 ne 27617 06 14 5 som _16 00 218809 95 274 5602 0 62724996 2 25E 02 C19 H17 N3 O17 52 1 02E 09 C6 H5 NO 3 29E 10 3 56E 11 7 90E 12 6 76E 11 z I7 2 2 L is aa 16 se 49757 84 15
23. 71 2 61E 01 1 12E 03 6 74E 03 5 38E 01 2 84E 02 3 75E 02 0 191515 449 9433 3 38E 01 3 38E 01 1 65E 05 5 66E 01 6 28E 01 2 34E 04 17 2916 447 972 1 72E 01 8 30E 02 2 37E 06 4 24E 01 4 52E 01 5 17E 05 0 08057 3 3 61E 01 3 61E 01 1 54E 05 5 75E 01 6 37E 01 2 32E 04 12 8760 ld p value cut off 11 0 05 lt lt Back Next gt gt Finish Cancel Comments 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 34 Mass Profiler Professional Application Guide Steps Detailed Instructions 7 Filter the remaining entities in your samples based on their relative abundance ratios among the samples and conditions Fold Change Step 7 of 8 a Review the summary plot b Move the Fold change cut off slider or type a value to change the Fol
24. 8 91 587161 06 242 1267 1 32204 12227325 60 98 15236 94 252 1223 1 41881 13849 57 2804 43 24664 38 iS 242 126 1 4454 1478 43 8 07 19956 94 S 118 0273 1 56415 334 19 36397 56 _ 8381 07 Z 331 1743 1 66055 8232 67 32506 03 28187 15 2 135 0546 1 61438 490 03 70839 23 23247 35 5 269 1856 1 72810 193083 66 122885 06 101919 17 1 amp 367 0879 1 76648 672 31 59115 54 15711 35 a 288 0846 1 78713 44201 70 120899 09 146429 75 E 624 2318 1 81863 197 73 5308 33 8925 63 z 126 03 18 1 78783 1 00 214704 69 11275 41 214 0742 1 79911 4887 56 78693 66 9760 13 548 2222 1 829148 637 15 35214 64 24873 97 574 1924 1 85713 360 52 2256 98 19865 22 260 1382 1 8685 700760 50 13481 28 6494078 50 131 0947 1 86945 6482 54 1349642 00 12041 04 833 3236 1 87342 8376 89 151452 67 16551 53 818 3584 1 87566 5 96 52030 17 7166 07 802 3822 1 87385 1 00 79652 98 22971 12 243 1114 1 87519 1144 79 624644 56 597 90 836 3311 1 87033 1 00 23352 98 1397 20 558 2223 1 88261 234 98 78765 70 33500 14 321 1209 1 90229 901 40 27423 53 15852 96 624 2328 1 91370 1534 27 4867 25 12230 00 0 20 50 100 226 0952 1 948577 8562 92 55808 46 8312 50 208 0846 1 94789 16601 41 49008 41 17250 61 Numeric Parameter 250 0482 2 03421 1 00 182690 02 76275 15 Fold change cut off _ 2 0 Minimum no of pairs 1 X Control Group f0 v Comments The Fold Change workflow step
25. Signal columns with non specific column headers and only numeric values in each column Please note that Mass Profiler Professional recognizes only the named required columns RT Mass Compound Name Formula and CAS ID plus these optional columns KEGG ID ChEBI ID HMP ID Lipid ID NCBI gi ID and Swiss Prot ID Mass Profiler Professional Application Guide 7 Mass Profiler Professional identifies all other columns that contain numeric values as signal columns However you can clear any column that Mass Profiler Professional wrongly identifies as a signal column Only the protein entities with a valid UniprotKB Accession values specified in the Swiss Prot ID column are considered for mapping in pathway analysis UniprotKB Entry name is not considered for mapping A generic file can contain one or more samples and many such files can be used to create an experiment Given a set of generic files if the same sample name occurs multiple times in the same file or across files Mass Profiler Professional uses only the first instance in alphanumeric file name order of the sample Mass Profiler Professional supports Identified Unidentified and Combined Identified Unidentified types of experiment creation for Generic data Select one of these three experiment types during Experiment Description The first entry that appears in any one of the Compound Name CASID Swiss Prot ID and Formula columns in that order is used to name the e
26. argets and components discovered by AMDIS Combination of entries identified by and not identified by Compound column C Formula column D CASID column E 13 Import and organize your data After you set up your project and create an experiment the MS Experiment Creation Wizard Figure 4 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 preparation includes grouping filtering alignment normalization and baselining Flow Chart of the MS Experiment Creation Wizard Step 6 Experiment Grouping Step 1 Select Data Source Step 9 Sample Summary Step 2 Select Data to Import Step 10 Normalization Criteria skipped Step 5 Sample Reordering Step 11 Baselining Options Steps 3 amp 4 Goto Step6 Create an initial analysis Figure 4 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 Elms Experiment Creat
27. ce actually refers to the feature chromatographic area 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 Mass Profiler Professional Application Guide 23 Steps Detailed Instructions 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 Type 5 0 ppm and 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 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 Elms Experiment Creation Wizard Step 8 of 11 b x 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 specified level Retention time Correction Perform RT correction Maximum Allawed RT Shift fos Clo fo s min Mass Window 15 0 ppm o mba RT Correcti
28. 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 1 Summary Report 2 Experiment Grouping 3 Filter Flags 4 Filter By Frequency 5 QC on samples 6 Significance Analysis 7 Fold Change Filter By Frequency 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 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 Not Infected Hep lt lt Back Next gt gt Finish Cancel 32 Mass Profiler Professional Application Guide 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 5 of 8
29. d 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 oie Type Analysis Significance Testing and Fold Change Step 7 of 8 x Steps Fold Change Compounds that satisfy a Fold change cut off of 2 0 in at least one condition pair are displayed by default To apply the new Fold change cut off drag the Fold change cut off slider or input the new cut off value in the text 1 Summary Report box 2 Experiment Grouping 3 Filter Flags Displaying 1111 out of 1152 entities with Fold change cut off of 2 0 in 1 out of 3 condition pairs with 0 as the control condition 4 Filter By Frequency Compound FC q20 vs 0D FC q50 vs OD 122 048 1 0158621 140478 42 405 162 56 413820 50 anion seniples 165 0655 1 01961 1 00 195816 59 11905 aoe 211 0956 1 02707 _ 108115 49 3268 47 122800 28 150 089 1 0711905 1 03 67758 70 8700 19 416 2389 1 12220 10182 01 55103 52 23355 56 8 IDBrowser Identifica 307 084 1 2057666 60159 58 82893 98 24785 24 375 1482 1 22568 9024 33 108 20 563 87 384 1191 1 25989 14650 62 1230 68 6 92 870 3104 1 29493 13883 03 51645 19 18332 73 204 0902 1 32119 458053 53 30312
30. d tt s lt s AR Detailed Instructions f Type the value for your first grouping in the Assign Value dialog box For the Malaria Demo type Not Infected g Click OK h Click your replicate Samples that share the same second parameter value in your data For example 3 1_pH7_pos_01 3 2 pH7_pos_01 3 3 pH pos 01 3 4 pH pos 01 i Click Assign Value j Type the value for your second grouping in the Assign Value dialog box For the Malaria data type Infected k 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 m Review your entries and grouping assignment accuracy in the Add Edit Experiment Parameter dialog box n Repeat the value assignments for individual or multiple samples as necessary to make corrections or changes o Click OK when the grouping for this parameter name Is complete Mass Profiler Professional Application Guide Comments e In this example the samples are assigned parameter values representing the Infection parameter e The highlighted samples are assigned the value typed in the Assign Value dialog box 19 Steps Pl 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 click on the Assign Yalues button and enter the
31. ed 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 rep 4 inthe Experiment Selection Dialog dialog box create a new experiment ElEzperiment Selection Dialog ox _cancel_ a b Click Create new experiment Click OK x 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 Open existing experiment Help 5 Inthe New Experiment dialog box enter and select information that guides your experiment creation 10 b Type a descriptive name for the experiment in Experiment name Select Mass Profiler Professional for Analysis type Select Unidentified or Combined Identified Unidentified for the Experiment type Select Analysis Significance Testing and Fold Change for Workflow type Type descriptive information in Experiment notes 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 can also create a new experiment in your pro
32. election Select 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 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 xi BOE a a a De Welcome to MassProfiler Pro ey project i Select what you would like to do from the options below then dick on OK After closing an open project you may create a new project from the to continue AE EE Menu bar click Project gt New OUER Project or from the Toolbar click Open recent project the New project button E Do not show this dialog again e Mass Profiler Professional Application Guide 9 Steps 3 Inthe Create New Project dialog box enter your project information Create New Project New Project Details Detail
33. eplicate 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 EE LL 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_O1 Infected Edit Parameter Delete Parameter lt lt Back Next gt gt Finish Cancel 20 Mass Profiler Professional Application Guide Steps 5 OPTIONAL Re order your 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 DE button 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 t
34. er By Frequency Displaying 8 sample s with 1 experiment parameter s To change use the button controls below 5 QC on samples 6 Significance Analysis 7 Fold Change 1 1_pH _pos_O1 Not Infected o 1 2_pH _pos_O1 Not Infected 8 IDBrowser Identifica 1 3_pH7_pos_01 Not Infected 1 4_pH _pos_O1 Not Infected 3 1_pH _pos_O01 Infected 3 2_pH _pos_01 Infected 3 3_pH _pos_01 Infected 3 4_pH _pos_O1 Infected Add Parameter Edit Parameter Delete Parameter Help lt lt Back Next gt gt Finish Cancel 3 Filter entities from your samples a Review the summary plot e A flag is a term used to denote the quality of an entity within a sample A flag indicates if the entity was b Click Re run Filter to enter parameters into the Filter Parameters based on the quality of their presence in specified samples and conditions Filter Flags Step 3 of dialog box detected in each sample as follows 8 c Mark the Present and Marginal check Present means the entity was boxes detected Absent means the entity x ee Acceptable Flags means the signal for the entity was lv Present saturated JV Marginal D i i Absent This filter removes irreproducible Retain Entities in which C atleast 100 0 of the values in any 1 out of 2 conditions have acceptable values at least 2 out of 8 samples have acceptable values Cancel entities from further con
35. fection Test Group Test ioi xi 1 1_pH7_pos_01 Not Infected Value 1 File Edit Format View Help 1 2_pH7_pos_01 Not Infected Value 2 Samples Infection Test Group 1 3_pH7_pos_01 Not Infected Value 3 Moh INLACTEG Vaie 1 4 pH _pos_01 Not Infected Value 4 Not Infected Value 3 1_pH7_pos_01 Infected Value 1 PH _pos_ Not Infected Value 3 1_pH7_pos_O1 Infected Value 3 2_pH7_pos_01 Infected Value 2 3 3_pH _pos_01 Infected Value 3 3 2_pH7_pos_O1 Infected Value 3 4 pH7_pos 01 Infected Value 4 hwuw 3 3_pH7_pos_O1 Infected value 3 4_pH7_pos_O1 Infected value 2 3 4 5 6 7 8 g MeN NN FY KF KF FE RIA 22 Mass Profiler Professional Application Guide Steps 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 Detailed Instructions 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 Filtering Filtering during the data import process may be used to reject low intensity data or restrict the range of data After data is imported there are several Filtering options that may be applied Filter by Frequency Abundance Variability Flags and Annotation For AMDIS experiments Number of ions filter is Number of Model io
36. ganize data A analysis Wes operations Set up a Do Significance Use the project and an Testing and Workflow experiment Fold Change Browser Import data Identify files into the features as experiment compounds Order and Save your group the project and data files experiment s Filter align and normalize Export your the sample project data Figure 2 Overview of the wizards that help you use MPP Where do get more information The Agilent workflow guides and overviews online Help and user guides provide you with additional detail techniques and explanations to perform advanced analysis operations e Agilent MassHunter Mass Profiler Professional User Manual You can find a PDF copy in the MPP installation folder C Program Files Agilent MassHunter Workstation Mass Profiler Professional docs manual e Agilent Metabolomics Workflow Discovery Workflow Guide p n 5990 7067EN Revision B e Agilent Metabolomics Workflow Discovery Workflow Overview p n 5990 7068EN Revision B e Class Prediction with Agilent Mass Profiler Professional Workflow Guide p n 5991 191 1EN e Class Prediction with Agilent Mass Profiler Professional Workflow Overview p n 5991 1912EN e MassHunter Profinder Quick Start Guide and online Help Mass Profiler Professional Application Guide 3 1 Prepare for your Experiment An experiment consists of the analysis of a set of replicate samples collected over a range of well defined para
37. he process for preparing for your experiment and performing an unbiased differential analysis is presented in the Metabolomics Discovery Workflow 5990 7067EN Mass Profiler Professional 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 For Agilent data you can use either MassHunter Profinder or the MassHunter Qualitative Analysis program to find features MassHunter Profinder To analyze Agilent LC and CE TOF Q TOF data in Mass Profiler Professional MassHunter Profinder is the preferred program to extract features from your sample data MassHunter Profinder is optimized for batch feature extraction and offers three different feature extraction workflows Refer to the MassHunter Profinder Quick Start Guide and online Help for details on the Mass Profiler Professional Supplemental disc MassHunter Qualitative Analysis The features in your sample data can be found and 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 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
38. ion Wizard Step 1 of 11 xi most important when you use the Pathway Analysis features of MPP Select Data Source Choose the data sources that will be used For the experiment e If you are importing a non Agilent data file click Generic MassHunter Qual MassHunter ICP MS C AMDIS Generic Organism Homo sapiens v 14 Cancel Mass Profiler Professional Application Guide Steps Detailed Instructions Comments 2 Select the molecular feature a Click Select Data Files e The file 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 e See Table 2 on page 12 for a comprehensive list of data sources you can select from based on your Select Data to Import Data may be imported from files or previous experiments exper me nt type e To control your progress through the wizard dialog boxes Click Next gt gt to go to the next Elms Experiment Creation Wizard Step 2 of 11 _ x step Click _ lt lt Back to return to prior lt lt Back Next gt gt Emish Cancel_ steps and make modifications to i your settings and previous b Select your samples in the Open entries dialog box If necessary browse to C Click Cancel to end the MS Program Files Agilent MassHunter Workstation Mass Profiler Professional sample
39. ject from the Menu bar Click Project gt New Experiment Toolbar Click the New experiment button al 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 Mass Profiler Professional Application Guide Steps new Experiment E E Experiment description Detailed Instructions Enter a name analysis type experiment type and a desired workflow type Analysis will guide you through a statistical significance test and Fold change analysis Data Import will guide you through experiment creation only Class Prediction will guide you through the creation and testing of a prediction model using imported training data Experiment name Analysis type Experiment type workflow type Experiment notes Malaria LEMS ESI pH 7 Cancel Comments e Table 1 on page 12 and Table 2 on page 12 show the selection and entry options available to you for the New Experiment
40. meter 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 Elorder Parameter Yalues f 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 appropriate icon Parameter Values e An example experiment grouping file that is in the Malaria Demo directory named MALARIA EXPERIMENT PARAMETERS to be loaded from file tsv e The tsv file is organized using tab separated values tsv that may be created edited and viewed using Microsoft Excel or Notepad Mass Profiler Professional Application Guide 21 Steps Detailed Instructions Comments c Load your experiment parameter e Creating and editing experiment grouping values from a sample file if parameter groupings may be more applicable by clicking the Import convenient for you using Microsoft parameters from samples zg button Excel Save your file as a tsv file ira id Malaria Application tsv Microsoft Excel 0 x File Home Insert Page Form Data Revie View Addl Y Qo 2 fe Samples B E D In
41. meters 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 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
42. mples 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 25 Steps 10 Select whether to normalize the data to reduce the variability caused by sample preparation and instrument response in the MS Experiment Creation Wizard Step 10 of 11 Detailed Instructions Comments a Select None for the Normalization e You may use normalization and Algorithm external scalar techniques to b Clear the Use External Scalar check reduce the variability in your data box that was caused by sample c Click Next preparation and instrument response Elms Experiment Creation Wizard Step 10 of 11 3 x Normalization Criteria
43. n the sample order to arl as rae polar use the appropriate buttons on the right to move samples up or down This sample y our s t a rti n g p oi n t wh en thi SS t e p Deselect the samples that need not be imported was be g un Seet Sample Name xI 1 1_pH _pos_01 1 3_pH _pos_01 1 4_pH _pos_01 3 1_pH _pos_01 3 2_pH _pos_01 3 3_pH _pos_01 3 4_pH _pos_01 qaaa Select All Unselect All lt lt Back Next gt gt Finish Cancel Mass Profiler Professional Application Guide 17 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 a Click Add Parameter Elms Experiment Creation Wizard Step 6 of 11 i 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 O experiment parameter s To change use the button controls below 5S BEE 1_pH _pos_01 l 2_ H _pos_O1 3_pH _pos_01
44. ng and Fold Change Step 6 of 8 Control Group infected X E x Steps 1 Summary Report 2 Experiment Grouping 3 Filter Flags 4 Filter By Frequency 5 QC on samples 6 Significance Analysis 7 Fold Change 8 IDBrowser Identifica Significance Analysis Entities are filtered based on their p values calculated from statistical analysis To apply the new p value cut off drag the p value cut off slider or input the new cut off value in the text box You will not be able to proceed to the next step if no entities pass the filter Displaying 271 out of 1220 entities satisfying corrected p value cut off 0 05 Multiple Testing Correction Test Description Selected Test 2way ANOVA p value computation Asymptotic Benjamini Hochberg EICI Result Summary pal p lt o os p lt o o2 P lt o 01 P lt 0 0050 P lt 0 0010 Corrected p value Infection 1220 75 67 61 38 35 Corrected p value Infection Treatme 1220 __ _ 49 44 36 32 _ 30 Corrected p value Treatment 1220 238 173 162 144 95 Expected by chance 3 1 0 0 0 p Corr Infection p Corr Infection Treatment 75 entities 49 entities 7 0059 3 74E 01 3 74E 01 1 60E 05 5 83E 0
45. ns Abundance filtering J Minimum absolute abundance 5000 counts compounds 7 Minimum relative abundance Yo Retention time filtering Limit to the largest JV Use all available data Min ETIO 0719 10 0719 Max RT 15 4101 15 4101 Number of ions Minimum number of ions Fa Single ion compounds only Mass Filtering Use all available data Min Mass 50 0167 50 00 Max Mass 2589 16 1000 Charge states C All charge states permitted Multiple charge states required lt lt Back Next gt gt Finish Cancel Mark the Use all available data check box under Retention time filtering 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 Click the Minimum number of ions button and type 2 under Number of ions Click Multiple charge states forbidden under Charge states Click Next Comments 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 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 abundan
46. ntity If no entry appears in any of these four columns the compound is considered to be unidentified 410 0033 0 298625 43892 34 352 0715 0 3075 Griseofulvin 126 07 8 41515 37 693 9807 0 32 237861 282 695 9493 0 32025 112335 9C 433 9571 0 318625 126290 125 611 9776 0 321375 391212 43 529 9748 0 321375 815284 791 9571 0 321875 184395 171 541 0607 0 419 cyclic adenosine diphosphate ribose 119340 53 3 427 0293 0 45175 Zidovudine diphosphate 106060 89 3 265 9593 0 74725 2 3 Diphospho D Glyceric Acid 138 81 8 559 0709 0 929 N1 5 Phospho D ribosyl AMP 1109 75 7 427 0292 0 936 Zidovudine diphosphate 0 936 106060 89 3 122 048 1 066625 Niacinamide 98 92 0 573554 346 1166 5 071375 Nifedipine 21829 25 4 95548 332 0699 6 865624 Fluorescein 22123 418 2698 10 325 Simvastatin 79902 63 9 174 1119 0 358 Arginine 74 79 3 143150 348 0486 0 575 Inosine 5 monophosphate IMP 131 99 7 624724 344 1836 8 48075 Granisetron metabolite 1 81795 Figure 3 Example spreadsheet to import non Agilent data Click this image to open the spreadsheet file for use as a template 8 Mass Profiler Professional 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 S
47. o prior steps in the workflow to improve the results 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 parameters To identify the Entities that passed the fold change cut off with IDBrowser click on the IDBrowser Identification button Identify Entities with IDBrowser IDBrowser Identification Compound Regulation 649 9687 0 31575 4 03E 10 1 19E 11 down 16 00 18910 66 14 21 2 693 9807 0 32 1 29E 03 9 68E 05 up 3 08 3 08 1 62 433 9571 0 318625 3 48E 03 _2 89E 04 up 216 2 16 EEN 611 9776 0 32137498 6 28E 04 4 65E 05 up 2 60 2 60 1 38 529 9748 0 32137498 2 52E 03 2 01E 04 up 2 49 2 49 1 32 791 9571 0 321875 4 07E 04 2 98E 05 up 2 63 2 63 1 39 633 9917 0 31725 1 30E 11 5 66E 14 down 16 00 34984 03 15 09 531 9423 0 31775 5 31E 11 6 38E 13 down 16 00 121746 46 16 89 447 972 0 322 1 79E 03 1 37E 04 up 2 01 2 01 1 00 793 9276 0 31875 3 26E 09 1 48E 10 down 16 00 41750 41 15 35 479 9197 0 32762498 4 63E 02 5 30E 03 down 2 18 2 18 1 13 217 9361 0 328625 2 31E 02 2 34E 03 down 2 00 2 00 1 00 397 9163 0 32937503 3 91E 02 4 3 1E 03 down 2 93 2 93 1 55 593 8678 0 32750002 1 30E 11 4 54E 14 down 16 00 101585 82 16 63 315 9133 0 330125 1 61E 02 1 58E 03 down
48. omatically begins identifying your entities and shows a progress bar Mass Profiler Professional Application Guide 39 Steps Detailed Instructions Comments h Review and make adjustments to the entity identifications as necessary using the ID Browser interface i 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 ioj xi File Edit View Identification Method Configuration Help i o P Run ID Wizard ale He amp abe Save and Return Il MS Spectrum Resuks x ik MS Peaks One MFE Spectrum 0 316 min x amp Structure Viewer x 2 oe tf Q 2 f mz 2 Abund Abund Norm zZ Sat No data to display 650 9758 554 Mat F e o CS 5 E a a 45 4 35 3 25 2 ess 1317 9713 1 2M NH4 0 5 700 800 900 1000 1100 1200 1300 Counts vs Mass to Charge m z LMS Spechu Rests Ga Compound List x if Cpd Te Label VY Name 4 Formula Y Score Y Mass Y AvgMass Y Std Dev Y Mass DB 2 gt 1 Cpd1 C19H18 N6 01055 C19 H18 N6 01 2 Cpd 2 C23 H10 N4 022 0 3 C23 H10 N4 022 66 93 693 9802 3 Cpd3 C14 H6 N6 05 53 0 C14 HE N6 0553 74 59 433 9564 o 4 Cpd 4 C24 H8 N2 018 0 321 C24 H8 N2 018 66 68 611 9779 5 Cpd 5 C13 H22 012 5 0 3 C13 H22 01255 80 529 9713 i 6 Cpd 6 0 322 791 9571 7 Cpd 7 C20 H15 CI N4 016
49. on 1 2_pH _pos_01 cef 1 3_pH _pos_01 cef eu als sad e You can review and make changes 3 1_pH _pos_O1 cef i 3 2_pH _pos_O1 cef to your selection during the next ila step before finalizing the experiment creation 3 4_pH _pos_O1 cef i Select Samples Remove lt lt Back Next gt gt Finish Cancel e A progress indicator is shown while your files are imported into MPP Progress Parsing Samples 3 8 Cancel 16 Mass Profiler Professional Application Guide Steps Detailed Instructions Comments 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 Click the Up 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 You may select a continuous range to import into your experiment of files with a click on a first file e Click Next and a Shift click on a last file that includes the range of files you want to select M5 Experiment creation wizard sten sord Click the Restore button at any Sample Reordering time to retur
50. on Method f Without Standards C With Standards fio of Internal Standards Pes RoTiiminutes mass Da Compound alignment RT Window fo Yo jo 1s min Mass Window 5 0 ppm 2 0 mDa Cancel 24 Comments e This step is omitted when the experiment type Is identified e 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 e lf 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 e 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 e Retention alignment rewrites the retention times in the data file Mass Profiler Professional Application Guide Steps Detailed Instructions 9 Review the compounds present and absent in each sample in the MS Experiment Creation Wizard box Step 9 of 11 c Click Next Q MS Experiment Creation Wizard Step 9 of 11 Xm Sample Summary From the Entities tab use merging o to manually merge er aE pectra of si re peti he e displayed to help merging cme Pa EE et Baan ncy of aligned cor as all the Mass vs tab di
51. or ee Agilent G3835AA MassHunter Mass 0909 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 9 4 Create your Initial Analysis 28 5 Save your project 43 6 Perform Advanced Operations 45 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 Agg Agilent Technologies Where is MPP used in your experiment MPP is
52. ow the steps in Define the with two parameter values must be replicate structure of your sample grouping with respect to the assigned experiment Experiment Grouping independent variables and the e An independent variable is an Step 2 of 8 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 replicate structure of your experiment in the MS Experiment Creation Wizard Step 6 of 11 on page 18 c Click Next when you have completed your experiment grouping el workflow Type Analysis Significance Testing and Fold Change Step 2 of 8 i xj Steps 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 1 Summary Report 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 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 2 Experiment Grouping 3 Filter Flags 4 Filt
53. s Malaria Demo for the Malaria Demo c Click the sample molecular feature data files to import into the experiment The example Malaria data files are 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 Experiment Creation Wizard without saving You can select a continuous range of files with a click on the first file and press Shift and click on the last file that includes the range of files you want to select You may select discontinuous individual by pressing Ctrl and clicking on additional files Mass Profiler Professional Application Guide 15 3 Import and Organize your Data Steps Detailed Instructions Comments A x Look in rr Malaria Demo b Ba 7_pos_O1 cef Recent Items My Documents 7 Ly Computer I File name bos_O1 cef 3 3_pH _pos_O1 cef 3 4_pH _pos_O1 cef Open e Network Files of type MassHunterQual Combined CEF X Cancel d Click Open to load the selected files Replicate samples are from the collection of multiple identical e Click Next eon samples from a population When Elms Experiment Creation Wizard Step2of11 __ es replicate samples are evaluated a Select Data to Import Data may be imported from files or previous experiments result IS obtained that more closely approximates the true value of the 1 1_pH _pos_01 cef populati
54. sideration by your analysis 30 Mass Profiler Professional Application Guide Steps Detailed Instructions d g h Ll workflow Type Analysis Significance Testing and Fold Change Step 3 of 8 Clear the Absent check box This flag is useful when you want to identify missing entities in the sample data Click at least _ s out of X samples have acceptable values The X is replaced in your display with the total number of samples in your data set Type 2 in the entry box By setting this parameter to a value of two or more one hit wonders are filtered Click OK Review the profile plot You are encouraged to repeat the Re run Filter until you obtain the best results for your experiment Click Next Comments The number of entities displayed above the profile plot is expected to decrease as you progress through the workflow A one hit wonder is an entity that appears in only one sample is absent from the replicate samples and does not provide any utility for statistical analysis Steps 1 Summary Report 2 Experiment Grouping 3 Filter Flags 4 Filter By Frequency 5 QC on samples 6 Significance 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
55. splays a scatter plot of compounds and spre pinin et has the si snag Se EN nds present or absent in individual samples Export For Recursion Total number of Aligned Compounds 4000 Retention Time Frequency 158 969 i ua 0 134 E B 60 1684 0 133 50 1684 0 133 1 144 9588 0 14366667 144 9588 0 144 3 114 9486 0 14 114 9486 0 143 2 232 9753 0 14733334 232 9753 0 147 6 204 9804 0 1455 204 9804 0 146 4 ER 9508 0 14366667 131 9508 0 144 3 172 9538 0 14649999 172 9538 0 146 6 120 0446 0 147 120 0446 0 147 2 237 0059 0 14356667 I 237 0059 0144 3 288 0461 0 272 288 0461 0 272 1 69 9938 0 274 69 9938 0 274 2 220 0447 0 276 220 0447 0 276 1 96 9908 0 27633336 36 9908 0 276 3 230 1009 0 27539998 230 1009 0 275 5 Legend Composite Spectra E 158 9694 0 13366668 Composite Spectra 19 000 18 000 17 000 sal Q MS Experiment Creation Wizard Step 9 of 11 Sample BUERNE From the ies tab use merging options to manually entities Spectra of selected entities Cp nd ie re D displays the frequency of ali eh palate across all the samples d catter plot of compounds and spreadsheet has the summary of aligned compounds present or absent in individual samples re displayed to help merging Export For Recursion Total number of Aligned Compounds 4000 Entities Compound Frequency Mass vs RT
56. t Grouping 3 Filter Flags 4 Filter By Frequency 5 QC on samples 6 Significance Analysis 7 Fold Change 8 IDBrowser Identifica Summary Report The distribution of normalized intensity values across all samples is displayed in the Profile Plot MassHunterQual IDENTIFIED_UNIDENTIFIED_COMPOUNDS experiment No of samplefs 8 Yv 4 L aw E 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 Invert Row Selection lt Back Next gt gt Finish Cancel Clear Row Selection Limit To Row Selection Select Columns Invert Column Selection Clear Column Selection Reset Filters Freeze Columns Before Unfreeze Columns Selection Mode Zoom Mode Invert Selection Clear Selection Limit To Selection Reset Zoom Copy View Export Column to Dataset Ctrl C Copy Print Ctrl P Copy View Ctrl C SEE gt Print Ctrl P Trellis Publish Catview Export As b Color By Venn Properties Ctrl R Properties Ctrl R Mass Profiler Professional Application Guide 29 Steps Detailed Instructions Comments 2 Define or adjust the sample a Click Add Parameter to define or e Note in order to proceed to the grouping with respect to the adjust your experiment grouping next step at least one parameter independent variables and the b Foll
57. the current view T aT Legend Profile plot Log2 Normalized MeSH Network Builder olor By 1 1 Control 000 Log2 and a memory monitor Click the aint Utilities a 19 0 18 1 1_Contr 1 2_Con 1 3_Con 1 4_Con 3 1_Con 3 2_Con 3 3_Con 3 4_Con 2 1_infe 2 2_Iinfe garbage can to reduce meman tetke Remove Entities with missing signal values Description Sa Analysis Significance Testing and Fold Change Eaanches on kternterations AI Sapia ees Class Prediction Build and Test Model Displaying 4414 0 selected ll l 110m of 138m f 42 Mass Profiler Professional 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 with a 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 your project choose Experiments a 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 Ix x Save in K Malaria Demo bd
58. tup the parameters and values for your database search N Compound Identification Wizard xj Compound Identification Browser Please set parameters for identification techniques M Identification method C MassHunter Methods B 05 00 Default m e EJ Identify Compounds Search Database t ee eeeese Generate Formulas C Mass and retention time retention time optional C Mass and retention time retention time required Match tolerance Mass 5 00 pom Retention time fo 100 minutes Help lt lt Back _ New Finish Cancel Search Criteria C Database Peak Limits Positivelons M Database selection Database path C MassHunter PCDL defaut csv a Search Criteria M Spectrum peak searches Maximum number of peaks to search when 5 peaks are not specified graphically 5 m x M Charge states if not known m Aggregates Tl Dimers e g 2M H Charge state range fi I Times eg 3M H Mass Profiler Professional Application Guide 37 4 Create your Initial Analysis Steps Detailed Instructions Comments 38 Mass Profiler Professional Application Guide 4 Create your Initial Analysis Steps Detailed Instructions Comments Ei compound Identification Wizard Te gt 2 g Click Finish when you have the method set up for your experiment ID Browser aut
59. 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 6 Mass Profiler Professional Application Guide 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 Non Agilent Data You can use Mass Profiler Professional to process non Agilent data Once imported into Mass Profiler Professional you can do statistical analysis and visualizations on non Agilent data in the same way that you analyze Agilent data except you are not able to do Spectral visualization Compound identification using ID Browser Create a recursion list for further mining of the data once interesting features are identified Create an MS MS inclusion list 1 Use your non Agilent data acquisition and analysis program to extract the features in your sample data 2 Export your non Agilent data to a spreadsheet file in comma separated tab separated or Excel xls and xlsx format Make sure that these required columns exist in the spreadsheet in this order RT Mass Compound Name Formula CAS ID One or more
60. will treat all compounds equally regardless of their intensity Options C None 2 Transform Baseline to median of all samples Baseline to median of control samples Finish Cancel Comments e There are four baselining options None Recommended if only a few features in the samples exist Z Iransform 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 Baseline to _ of all samples The abundance for each compound is normalized to its selected statistical abundance across all of the samples This has the effect of reducing the weight of very large and very small compound features on later statistical analyses Baseline to _ of control samples The abundance for each compound is normalized to its selected statistical abundance across just the samples selected as the control samples This has the 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 features If you selected Analysis Significance Testing and Fold Change for the Workflow type in the New Experiment dialog box you immediately begin your analysis Mass Profiler Professional Application Guide 27 4 Create your Initial Analysis 28 The Analysis Significance Testing
61. wo parameters will be used For analysis in the quided 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 3 experiment parameter s To change use the button controls below bb Ob of O Samples 1 1_pH _pos_O1 iNot Infected 3 1_pH7_pos_O1_ _ infected 1 2_pH _pos_O1 Not Infected 3 2_pH _pos_01 Infected 1 3_pH _pos_01 Not Infected 3 3_pH _pos_O1 Infected 1 4_pH _pos_01 Not Infected 3 4_pH _pos_O1 Infected Add Parameter Edit Parameter Delete Parameter lt lt Back Next gt gt Finish Cancel 6 OPTIONAL Saving and importing experiment grouping information in a spreadsheet 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 Sal 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 J Comments When you have more than one parameter associated with your samples each para
62. y 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 Mass Profiler Professional Application Guide Steps Assign Yalue x Enter a value for the selected samples Not Infected ce 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 x 1 1_pH7_pos_01 1 2_pH7_pos_01 1 3_pH7_pos_01 1 4_pH7_pos_01 Not Infected Not Infected 3 3_pH7_pos_01 3 4_PH7_pos_01 x Enter a value for the selected samples infecte

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