If you are unfamiliar with the Thermo Compound Discoverer™ 2.0 application, follow the topics in this tutorial to set up a study and an analysis, process a set of example Xcalibur™ RAW files, review the result file produced by the analysis, and export the results to a Microsoft™ Excel™ spreadsheet.

GettingStarted

To create a practice study, use the example Xcalibur RAW files that are provided on the software installation media. You can find these files on the Compound Discoverer USB key in the following folder:

Example Studies\Metabolomics Studies\ZDF\

Copy the ZDF folder to your data processing computer. This figure shows the tutorial’s workflow.

Compound Discoverer 2.0 Metabolomics Tutorial

Contents

• Getting Started

• Starting the Application

• Checking the Computer’s Access to the External Databases

• Setting Up a New Study and a New Analysis

• Submitting the Analysis to the Job Queue

• Reviewing the Analysis Results

• Exporting the Results to an Excel Spreadsheet

Start the Compound Discoverer application and check the computer’s access to the external mass spectrometry databases.

Open the result file and review, filter, and sort the data.

Export the results to an Excel spreadsheet.

Confirm the analysis and start the run.

Use the New Study and Analysis Wizard to do the following:1. Create a new study and select a processing workflow.2. Add the files that you want to process to the study.3. Define and assign the study factors for the sample set.4. Set up the sample groups and ratios for the analysis.

Start the Compound Discoverer application.

Check the computer’s access to the KEGG™ Pathways, mzCloud™, and ChemSpider™ databases.

© 2015 Thermo Fisher Scientific Inc.All rights reserved.

Revision A XCALI-97803

Starting theApplication

To start the application• From the taskbar, choose Start > All Programs (or Programs) > Thermo Compound Discoverer 2.0.

–or–

• From the computer desktop, double-click the Compound Discoverer icon, .

The application opens to the Start page.

Checking theComputer’s

Access to theExternal

Databases

This tutorial uses a processing workflow that runs mzCloud, ChemSpider, and KEGG Pathways searches. To run the searches, your processing computer must have unblocked access to these databases on the Internet.

To verify that your computer has access to all three external databases1. From the menu bar, choose Help > Communication Tests.

The Communication Tests window opens to the KEGG Pathways page.2. Run the KEGG Pathways communication tests by clicking Run Tests. When the tests are complete, go to the

next step.3. Run the mzCloud communication tests as follows:

a. Click the mzCloud tab. b. Click Run Tests. When the tests are complete, go to the next step.

4. Run the ChemSpider communication test as follows:a. Click the ChemSpider tab. b. Click Run Tests.

If your computer has an Internet connection, but one or more of these tests fails, contact your IT department. If your computer does not have an Internet connection or one of the communication tests fails, you can complete this tutorial by using the defined metabolomics processing workflow that does not include an external database search.

Go to the next topic “Setting Up a New Study and a New Analysis.”

Setting Up aNew Studyand a New

Analysis

Make sure to copy the Xcalibur RAW files from the Compound Discoverer 2.0 media to an appropriate folder on your processing computer. See “Getting Started” on page 1.

Follow these topics to create a new study and a new analysis:1. Setting Up the Study Folders2. Selecting the Processing Workflow3. Adding the Input Files to the Study

Menu bar Toolbar

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4. Defining the Study Factors5. Setting Up the Sample Groups and Ratios6. Modifying the Processing Workflow

Setting Up theStudy Folders

Each time you create a new study, the application creates a new study folder with the same name and stores the study file (.cdStudy) in the new folder. When you first install the Compound Discoverer application, you must set up a top-level folder for the study folders.

To name the new study and set up the top-level folder

1. From the menu bar, choose File > New Study and Analysis.

The New Study and Analysis Wizard opens.2. Click Next to open the Study Name and Processing Workflow page.

The first time you open the wizard, the top-level folder for storing the study folders is undefined.

3. In the Study Name box, name the study.

• For a metabolomics study with the ZDF (rat) example files, type ZDF Study.

• For a study that includes your own Xcalibur RAW files, type an appropriate name.4. Select the folder where you want to store your Compound Discoverer study folders as follows:

a. Click the browse icon, , next to the Studies Folder box.b. Browse to your local disk drive or a location on your local network. c. Click New Folder to create a new folder and name the folder Studies.

After you select or create a top-level folder, the Next button becomes available. Do not click Next. Stay on this wizard page and go to the next topic, Selecting the Processing Workflow.

When you complete the wizard, the application creates the ZDF Study.cdStudy file, stores the study file in the ZDF Study folder, and stores the ZDF Study folder in the Studies folder. When you run an analysis, the application stores the result files (.cdResult) in the ZDF Study folder.

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Selecting theProcessing

Workflow

In the Compound Discoverer application, the processing method that interprets the raw data is called a processing workflow (.cdProcessingWF). The application provides defined processing workflows for several applications including metabolomics experiments. This tutorial uses a defined processing workflow that searches the mzCloud, ChemSpider, and KEGG Pathways databases to identify the compounds detected in the input files (Xcalibur RAW files).

To select the processing workflow1. Under Processing, select the following processing workflow from the Workflow list:

Workflow Templates\Metabolomics\Untargeted Metabolomics workflow with statistics and ID using mzCloud ChemSpider KEGG Pathways

A description of the processing workflow appears in the Workflow Description box.

2. Read the description.

3. Click Next to open the Input File Selection page of the New Study and Analysis Wizard.

Adding the InputFiles to the Study

To add input files to the study1. On the Input File Selection page of the New Study and Analysis Wizard, click Add Files in the command bar.

The Add Files dialog box opens.2. Browse to the folder where you copied the Xcalibur RAW files.3. Select all of the Xcalibur RAW files in this folder and click Open.

4. Click Next to open the Input File Characterization page of the wizard.

Note If your computer does not have an Internet connection or fails one or more of the communication tests, select the following processing workflow:

Workflow Templates\Metabolomics\Untargeted Metabolomics workflow with ID using internal database only

This processing workflow does not search any of the external databases.

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Defining theStudy Factors

This figure shows the newly added samples in the Samples area. By default, the application assigns Sample as the Sample Type to new samples. To group the samples according to your experimental design, you must define the study factors. In this tutorial, you are comparing the compounds in the plasma from two strains of ZDF rats. The phenotypes for the two strains are Fatty and Lean.

To define the study factors, follow these procedures:1. To select the delimiters that separate the factors in the file names2. To set up Phenotype as a categorical study factor3. To assign the phenotypes and the Blank sample type

To select the delimiters that separate the factors in the file names 1. Select the Underscore check box.

In the example files, the underscore character is the delimiter that separates the study factors from the other parts of the file name.

The study factor is Phenotype (Lean or Fatty).

To set up Phenotype as a categorical study factor1. In the Study Factors area of the Input File Characterization page, choose Add > Categorical Factor.

ZDF_Fatty_1 ZDF_Fatty_2 ZDF_Fatty_3

ZDF_Lean_1 ZDF_Lean_2 ZDF_Lean_3

Default Sample Type

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The categorical study factor editor opens in the Study Factors area with the [new factor] box selected.

2. Type the factor name: Phenotype.

If the editor closes before you type Phenotype, click Edit to reopen it. Select [new factor] and type Phenotype.

3. To add the Fatty phenotype to the Items list, do the following:a. In the box next to the Add and Delete buttons, type F.

The application parses the file names in the Sample Identifier column and Fatty appears in the box.b. Click Add.

4. To add the Lean phenotype to the Items list, do the following:a. Type L in the box.b. Click Add.

5. Click Apply.

The editor closes and the Edit button replaces the Apply and Cancel buttons.

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To assign the phenotypes and the Blank sample type1. In the command bar next to the delimiters, click Assign.

The application does the following:

• Assigns the study factor items for the Phenotype study factor, Fatty or Lean, to the samples.

• Assigns the Blank sample type to the Blank.raw file.

This figure shows the study variable assignments. Study variables include the study factor and sample type assignments for the samples.

Because the file names for the Pooled_ddMS2_Top3 sample and the Blank sample do not match the defined pattern, ZDF_categorical factor_, the application assigns these samples a value of n/a for the Phenotype study factor. The n/a assignment is correct, as neither sample belongs to a phenotype group (Fatty or Lean).

2. Click Next to open the Sample Groups and Ratios page of the wizard.

Setting Up theSample Groups

and Ratios

Use the Sample Groups and Ratios page of the wizard to set up a differential analysis for the two phenotypes: Lean and Fatty.

Note The Pooled_ddMS2_Top3 sample is a blend of the six non-blank samples. The Pooled_ddMS2_Top3.raw file contains the data-dependent MS/MS fragmentation scans for the input file set. The Blank sample is a matrix blank and only contains the background compounds.

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To set up the sample groups and ratios1. In the Study Variables area, select the Phenotype check box.

The sample groups—n/a, Fatty, and Lean—appear in the Generated Sample Groups area.

2. In the Bulk Ratio Generation area, select the Phenotype: Lean check box to make the Lean group the control group.

The Add Ratios button becomes available.3. Click Add Ratios.

The ratio, Fatty/Lean, appears in the Generated Ratios area.

The analysis is set up to compare the two sample groups: Fatty versus Lean.

4. Click Finish to save the study and close the wizard.

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The ZDF Study tab, the two analysis page tabs (Grouping & Ratios and Workflows), and the Analysis pane appear. The ZDF Study tab does not have an asterisk, which means that this new study has no unsaved changes.

The Analysis pane lists the eight input files. The analysis is set up to combine the mass spectral data in these files into one result file; that is, the As Batch check box is clear and the Result File name is available for editing.

Modifying theProcessing

Workflow

Before submitting the analysis to the job queue, review the processing workflow and make changes as needed.

To review the processing workflow1. Click the Workflows tab to open the Workflows page.2. In the Workflow Tree area, select the Detect Unknown Compounds node.

The Parameters page for the Detect Unknown Compounds node opens.3. Check the Min. Peak Intensity setting against the suggested setting for your data set.

The minimum peak intensity setting defines the base peak intensity for the unknown compound detection. For this tutorial, keep the setting of 2 000 000.

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Table 1 lists the recommended range for the minimum peak intensity parameter. The optimal setting depends on the sensitivity of the mass spectrometer.

4. In the Workflow Tree area, select the Search ChemSpider node.

The Parameters page for the Search ChemSpider node opens.

5. Select the databases that you want the analysis to search. Do not change the selection for this tutorial.6. Save the modified processing workflow as follows, click Save in the Workflows command bar.7. In the Save Workflows dialog box, rename the processing workflow, select the folder where you want to store the

workflow, and click Save.

Submittingthe Analysis

to the JobQueue

If the Run command on the Analysis pane is available, you are ready to start the processing run.

To submit the analysis to the job queue1. To create one result file for the input file set, leave the As Batch check box clear.

By default, the application uses the name of the first input file as the result file name.2. In the Result File box, rename the result file Fatty vs Lean.

3. Click Run to submit the analysis to the job queue.

The Job Queue page opens and an asterisk appears next to the study name. The asterisk indicates that you made unsaved changes to the study. In this case, you submitted a run, which adds the run to the Analysis Results page of the study. If you close the study before saving this change, the Analysis Results page does not list the run.

Table 1. Recommended minimum peak intensity range

Mass spectrometer Minimum peak intensity(chromatographic peak height)

Q Exactive™, Q Exactive Plus™, Q Exactive HF, Orbitrap Fusion™ 1 000 000 to 5 000 000

Exactive™, Exactive Plus™, Orbitrap Elite™, Orbitrap Velos Pro™ 100 000 to 500 000

Orbitrap XL, Orbitrap Velos 25 000 to 100 000

Result file name

Run command

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4. To view the processing messages, click the expand icon, , to the left of the job row.

This figure shows a partial list of the processing messages for the run.

5. Before or after the run is completed, save the study by choosing File > Save All in the menu bar. Leave the Job Queue page open and go to “Reviewing the Analysis Results.”

Tip Saving the link to the current analysis on the Analysis Results page saves you time when you want to rerun the current analysis or a modified version of the analysis.

Note During the run, the Align Retention Times node cropped out some of the data points in the input files to perform the chromatographic alignment. The yellow background for these messages (not shown in the following figure) alerts you to this modification, and when the run is completed, the Warnings status appears in the Details column. Cropping out data points that become negative (negative retention time) during the alignment process is relatively common. You can ignore these messages.

Study tab with an asterisk

Expand icon

Processing messages

Warnings status

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Reviewingthe Analysis

Results

Follow these topics to review the analysis results:

• Opening the Result File

• Showing or Hiding the Result Tables

• Showing or Hiding Table Columns

• Working with the Chromatograms View

• Working with the Mass Spectrum View

• Working with the Volcano Plot for the Differential Analysis

• Viewing the Principal Component Analysis

• Viewing the KEGG Pathways for a Compound

• Using the Result Filters View

• Saving the Result File Layout

For information about modifying the layout of the graphical views, refer to the following Compound Discoverer Help topic: Rearranging the Graphical Views.

For more information about a specific result table or view, select the table or view to make it active and press the F1 key. The Help topic for the active item opens. The Compound Discoverer application provides F1 Help for all of the views that you access from the View menu and all of the result tables.

Opening theResult File

You can open a result file from multiple locations: the Job Queue page, the Analysis Results page of a study, the Compound Discoverer Start Page, or the menu bar.

To open the result file generated by the analysis

When the run is completed, do one of the following:

• Double-click the run on the Job Queue page.

• Double-click the run on the Analysis Results page of the study.

• On the Compound Discoverer Start Page, do one of the following: Under Recent Results, click the Fatty vs Lean hyperlink.

Under What Would You Like to Do, click Open Result. Browse to the Drive:\Studies\ZDF Study folder, select the Fatty vs Lean.cdResult file, and click Open.

• From the menu bar, choose File > Open Result. Browse to the Drive:\Studies\ZDF Study folder, select the Fatty vs Lean.cdResult file, and click Open.

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The result file opens as a tabbed document in the application window. By default, the Chromatograms view opens in the upper left, the Mass Spectrum view opens in the upper right, and a set of tabbed tables opens in the bottom half of the page. Neither the Chromatograms view nor the Mass Spectrum view is populated with data.

This figure shows the default layout for the Fatty vs Lean.cdResult file.

Showing orHiding the

Result Tables

Table 2 describes the master result tables that the selected processing workflow produces. The Compounds table is the active table and is sorted by area. The first row displays the compound with the largest chromatographic peak area (found in one of the input files). Because the selected processing workflow includes the Mark Background Compounds node, the Compounds tab has a filter icon with a check mark ( ). The compounds that the analysis identified as background compounds are marked as background compounds in both the blank and non-blank samples and are hidden from the table.

The default layout does not include all of the results tables that the selected processing workflow produces. By default, the Filled Gaps related table produced by the Fill Gaps node is a hidden table in the first set of related tables.

Result Table Chooser icon

Field Chooser icon Click to open the tabbed related tables.

Table 2. Master tables for an untargeted workflow that searches the mzCloud, ChemSpider, and KEGG Pathways databases

Result table Description

Compounds Lists all of the compounds that the analysis detected grouped by their molecular weight and retention time (MW x RT) dimensions across all of the input files.

Compounds per File Lists all of the compounds that the analysis detected across all of the input files on a per file basis. Does not list compounds that the Fill Gaps node detected by filling a full gap.

Features Lists all of the features (ions with the same mass-to-charge values and retention time) that the analysis detected across all of the input files on a per file basis.

mzCloud Results Lists the mzCloud search results across all of the input files.

ChemSpider Results Lists the ChemSpider search results across all of the input files.

KEGG Pathways Lists the KEGG Pathways that contains at least one of the detected compounds across all of the input files.

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To show or hide result tables1. Click the Result Table Chooser icon, , to the left of the result table tabs.

The Result Table Chooser dialog box opens.

2. Select the check box that corresponds to the table that you want to show, or clear the check box that corresponds to the table that you want to hide.

By default, the Adducts and Filled Gaps tables are hidden.3. For this tutorial, select the Filled Gaps check box.4. Click OK.

Showing orHiding Table

Columns

Table 3 describes the Compound table columns that the selected processing workflow produces.

Table 3. Compounds table columns (Sheet 1 of 2)

Column or columns Descriptions

Name Displays the name of the compound identified by the mzCloud search.

Predicted Formula Displays the neutral chemical formula predicted by the Predict Composition node.

Molecular weight and RT (min) Display the neutral molecular weight and retention time of the selected compound.

Area (Max) Displays the area of the largest chromatographic peak detected for the selected compound (same MW×RT dimensions within the user-specified tolerances) across all of the input files.

mzCloud Results Displays the search results for the selected compound across all of the input files.

#Pathways Displays the number of KEGG pathways in the KEGG Pathways related table for the compound.

Pathways Indicates whether the compound in the selected row is present in the named pathway. A rectangle in the intersecting table cell for a compound and pathway indicates that the pathway contains the compound. To help you differentiate the pathway columns as you scroll the table, each pathway is represented by a different color.

mzCloud Best Match Displays the best match score from the mzCloud search for the compound.

#Adducts (hidden) Displays the number of adduct ions that the analysis found for the compound.

Background (hidden) When the Background filter is turned off, displays a clear or selected check box that indicates whether the compound is a background compound.

Area (hidden) Displays an area column for each input file.

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In the Compounds table, the following columns are hidden:

• #Adducts

• Area

• Background

• Gap Status

To display or hide a table column1. Click the Field Chooser icon, , to open the Field Chooser box.

2. Do one of the following:

• To display a column, select the column’s check box.

• To hide a column, clear the column’s check box.

To show the Area column in the Compounds table 1. Click the Field Chooser icon, .2. Select the Area check box.

The Area column appears in the Compounds table to the left of the Group Areas column.

Gap Status (hidden) Displays the gap status of the chromatographic peaks for the selected compound in each input file.

Group Areas, Group CV%, Ratio, Log2 Fold Change, P-Value, and Adj. P-Value

Display the statistical calculations of the Differential Analysis node.

Note None of the defined processing workflows include the Normalize Areas node. This node creates the Normalized Areas column, which is a hidden column in the Compounds table.

Table 3. Compounds table columns (Sheet 2 of 2)

Column or columns Descriptions

Close icon

Expand icon

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3. To close the Field Chooser box, click its close icon, .

4. To view the sample names for the area columns, click the expand icon, , to the right of the column name.

The names of the input files appear vertically above the individual area columns. The name of the first input file, blank.raw (F1), appears in bold font and has an asterisk to indicate that it is the selected area column.

5. Click the collapse icon, , to hide the sample names.6. To hide the Area column for the remaining portion of this tutorial, open the Field Chooser box and clear the

Area check box.

To view the tables related to the Compounds table 1. Click Show Related Tables at the bottom of the page to display the tables related to the Compounds table.2. In the related tables set, click the Compounds per File tab.

The rows are sorted by area.

The Blank sample is hidden. Study file F4 contains the largest amount of the selected compound by peak area; however, its Area value does not match the Area (Max.) value in the compounds table.

The Area (Max.) column displays the largest peak area for a compound across the input files. The peak area for an individual peak is the sum of the peaks for its related features. The features for study file F4 include the detected features (adduct ions) shown in the Features related table and the feature (gap-filled peak) shown in the Filled Gaps table.

Note If you leave the Field Chooser box open, it closes when you make another table the active table.

Collapse icon

Area column

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3. To understand the discrepancy in peak areas, do the following:a. Click Show Related Tables at the bottom of the page to show the tables related to the Compounds per File

table.b. Click the Features tab to display the Features table. Notice that the table lists two features and that the areas

of the features add up to the Area value in the Compounds per File table.

c. Click Hide Related Tables above the set of secondary related tables.d. In the first set of related tables, click the Filled Gaps tab to display the Filled Gaps table.

Notice that the Filled Gaps table lists a peak area for an ion, [M+Na]+1, that is not listed in the Features table. When you add this peak area to the peak area in the Compounds per File table, the sum equals the Area (Max.) value for the selected compound in the Compounds table.

Source of peak area Neutral MW RT or Exp. RT [min] Ion Area for study file F4

Compounds table Area (Max.) 202.0450 0.830 – 316 982 435

Compounds per File related table 202.0450 0.831 – 316 403 732

Features related table 202.0450 0.831 [M+H]+1 314 644 260

202.0451 0.829 [M+H–H2O]+1 1 759 472

Filled Gaps related table 202.0450 0.830 [M+Na]+1 578 703

Total area for the related features

316 982 435

Gap filled by a re-detected peak

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Working withthe

ChromatogramsView

To view a mass chromatogram1. In the Compounds table, click the Area (Max.) column heading to sort the table by Area (Max.) in descending

order. 2. Select the first row to display the overlaid traces for the compound with the largest peak area.

By default the display options for the Chromatograms view are set to Show ToolTips, Show Detected Peaks, Show Legend, and Zoom to Detected Peaks.

This figure displays the overlaid traces for the input files that contain the selected compound and the shortcut menu for the Chromatograms view. The Mass Spectrum view is closed.

3. Under Filter By, expand Phenotype, Sample Type, and Sample by clicking their expand icons, .

Expand icon

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4. Review the information in the collapsible left pane:

• Under Group By, the count for Phenotype is 3 out of 3 (3/3). Under Filter By, Phenotype lists three groups and all three groups are selected.

• Under Group By, the count for Sample Type is 1 out of 2 (1/2), and the count for Sample is 7 out of 8 (7/8). Blank is the missing sample type and the missing sample.

• Under Filter By > Sample Type, the Blank check box is clear (by default) and the Chromatograms view does not display the trace for the blank sample.

5. To remove traces for the n/a group in the Chromatograms view, under Filter By > Phenotype, clear the n/a check box.

6. To view the chromatographic traces without the shading that can mask underlying peaks, right-click the Chromatograms view and choose Display Options > Show Detected Peaks.

This figure shows the overlaid traces for the six samples that you are comparing. The application displays the two sample groups in different colors. The legend shows the display colors for the groups: green for Fatty and yellow for Lean. Under Group By, the count for Phenotype is now 2 out of 3, and the count for Sample is now 6 out of 8. Filtering out the n/a sample (Pooled_ddMS2_Top3) reduced both of these counts by one.

Working withthe Mass

Spectrum View

To view a mass spectrum for a compound with a predicted formula1. Sort the Compounds table by the mzCloud Best Match column in descending order.2. Select the second row in the Compounds table. Creatine is the selected compound.3. To expand the mass spectrum view to the full screen width, do one of the following:

• Close the Chromatograms view by clicking the Close, , icon on its title bar.

–or–

• Drag the Mass Spectrum view by its title bar to a second monitor.

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This figure shows the Mass Spectrum view as a floating window. The spectrum tree on the left contains full scans and data-dependent fragmentation scans. The spectrum on the right includes color-coded centroids, as the selected compound has a predicted formula.

The Mass Spectrum view shows the full MS scan that is closest to the retention time displayed in the RT [min] column.

The mass spectral peaks in the full MS scan are color-coded as follows:

• Lavender bars indicate centroids for A0 (monoisotopic) ions. The x-axis position and the width of the bar reflect the expected m/z value of the centroid and the user-specified mass tolerance, respectively.

• Green rectangles indicate matching centroids for isotopic ions. When you zoom in on the matching centroid, the x-axis position and width of the rectangle reflect the expected m/z value of the centroid and the user-specified mass tolerance, respectively. The y-axis position and height of the rectangle reflect the expected relative intensity of the centroid and the user-specified intensity tolerance, respectively.

• Red rectangles indicate centroids that are missing from the expected isotopic pattern.

• Blue rectangles indicate centroids that are missing from the expected isotopic pattern but that are also expected to have an intensity below the measured baseline noise [determined by the Fourier transform mass spectrometry (FTMS) mass analyzer].

To view the results of an mzCloud search for a compound and display a mirror plot1. With the table sorted by the mzCloud Best Match in descending order, select Acetyl-L-Carnitine (row 5) in the

Compounds table. 2. Click Show Related Tables to display the related tables below the master tables.3. Click the mzCloud Results tab to display the mzCloud Results table.4. Select the first row in the mzCloud Results related table.

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A mirror plot appears in the Mass Spectrum view with the mass spectrum from the Pooled_ddMS2 sample on the top and the mzCloud reference spectrum on the bottom.

Working withthe Volcano Plot

for theDifferential

Analysis

To view the volcano plot for the current sample comparison1. From the menu bar, choose View > Differential Analysis.

The Differential Analysis view opens as a docked view next to the result tables. You can move the view to other docked positions or move it to another monitor as a floating window. Figure 1 shows the Differential Analysis view as a floating window.

The view displays a volcano plot. A volcano plot is a plot of the p-value, the result of a significance test, on the y axis versus the log2 fold change between two sample groups on the x axis. By default, the analysis is set to a p-value of 0.05 on the y axis and a Log 2 Fold Change of 1 (two-fold change) on the x axis.

The y-axis scale is the –Log10 of the p-value. As the p-value increases from 0 to 1, the –Log10 of the p-value decreases from infinity to 0 (–Log10 0.05 equals 1.3).

2. To display the legend, right-click the view and choose Show Legend.

The legend appears at the bottom of the page. Each data point represents a compound. Placing the pointer over a data point displays a ToolTip with the coordinates, MW, RT, maximum area, and number of adducts.

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Figure 1. Differential Analysis view with the default settings for P-value and Log2 Fold Change

The compounds in the shaded regions are statistically significant for the specified p-value and outside the upper and lower fold change thresholds.

To change the p-value

Move the P-value slider to the left or right. Or, type a value in the box next to the slider.

To change the log 2 fold change value

Move the Log2 Fold change slider to the left or right. Or, type a value in the box next to the slider.

ToolTip

Legend

p-value of 0.05

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This figure shows a volcano plot for a p-value setting of 0.001 and a fold change of 3 (log2 3 = 1.6).

To select the compounds in the red region and check the selected points1. Drag the pointer across the red region to select it. Then release the mouse button.

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2. Right-click the selected region and choose Check All Visible Points.

The color of the checked points changes from red to blue and the application selects the check boxes for the selected compounds in the Compounds table.

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3.Double-click a compound in the volcano plot to view it in the Compounds table and the other graphical views that are open.

This figure shows the ToolTip over the selected compound in the volcano plot. The Chromatograms view displays the overlaid chromatograms for the compound, and the Mass Spectrum view displays the compound’s full MS scan.

4. To sort the table by two criteria (selected check box in the Checked column and MW), do the following:a. In the Compounds table, click the Checked column heading to sort the table by the checked column.b. Hold down the CTRL key and click the Molecular Weight column heading to sort the checked rows by

molecular weight. To sort the checked rows by MW in descending order, make sure that the sort icon is facing down .

This figure shows the Compounds table sorted by the Checked status and the molecular weight in descending order.

Selected compound

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Viewing thePrincipal

ComponentAnalysis

To view the Principal Component Analysis 1. In the menu bar, choose View > Principal Component Analysis.

The view opens to the Scores Plot page in the lower right of the result file page.2. If you have a second monitor, drag the view by its title bar to the second monitor.3. In the collapsible left pane, clear the check box for the n/a sample group under Filter By > Phenotype if it is not

already cleared.

Use the scores plot to interpret the relationship among the sample groups. Sample groups that are near each other are similar.

Viewing theKEGG Pathwaysfor a Compound

To view the KEGG pathways that include the compound1. In the master Compounds table, sort the table by the Area (Max.) column in descending order.2. Select Creatine (row 7).3. Click Show Related Tables to display the first set of related tables.4. Click the KEGG Pathways tab to make it the active table and select the Arginine and Proline Metabolism

pathway (row 2).

5. In the menu bar, choose View > KEGG Pathways.

The KEGG Pathways view appears as a docked view.

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6. Drag the view by its title bar to a second monitor.

A blue circle represents the selected compound. Red circles represent the related compounds.

7. Place the mouse pointer over the blue circle to display a ToolTip and the compound’s structure.

Using the ResultFilters View

The analysis found 3074 compounds, including 1174 background compounds. Set up a filter that keeps the compounds of interest; that is, the compounds with a low p-value (statistically significant).

Placing the mouse pointer over the vertical scroll bar on the right displays a ToolTip with the number of compounds in the table. The table currently displays 1900 compounds.

To reduce the number of Compounds to review1. Click the Compounds tab in the set of master tables.2. From the menu bar, choose View > Result Filters.

Selected compound

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The Result Filters view opens as a floating window. Because the processing workflow included the Mark Background Compounds node, the filter for the Compounds table already includes a Background filter and the table does not display the background compounds.

3. In the right pane of the Result Filters view, set up a filter for the p-value as follows:a. Click Add Property and select P-Value from the list.

b. In the pink relation list, select Is Less Than or Equal To. c. In the value box next to the relation list, type 0.001.d. In the pink condition list, select In Ratio.e. In the Green sample list, select Fatty/Lean.

4. Click Apply Filters.

The filter reduces the number of displayed rows in the Compounds table from 1900 to 105.

Saving theResult File

Layout

If you close a result file without saving its modified layout, it opens with the default layout the next time you open it.

To save the layout

With the result page selected as the active page, click the Save the Currently Active Item icon, , in the toolbar.

To restore the default layout1. Open the ZDF Study folder on your processing computer.2. Delete the Fatty vs Lean.cdResultView file.

Tip Saving a result file saves the modified layout. To restore the default layout, open the study folder that contains the result file and delete the filename.cdResultView file.

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Exporting theResults to an

ExcelSpreadsheet

Before exporting the results to a spreadsheet, filter the results table as described in “To reduce the number of Compounds to review,” and sort the rows by p-value in ascending order.

To check the number of table rows

Place the mouse cursor over the vertical scroll bar to the right of the compounds table. A ToolTip appears with the row count.

To sort the rows by p-value in ascending order

Click the P-Value column heading until the table is sorted by p-value in ascending order.

To export the filtered and sorted results to an Excel spreadsheet1. Right-click the Compounds table and choose Export > Export to Excel.

The Export to Excel dialog box opens.2. Check the file name and location in the Path box. Change the file name and location as appropriate.3. In the Options area, select the Open File After Export check box.

4. Click Export.

The Excel spreadsheet opens.

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Trademarks The following are trademarks in the United States: Compound Discoverer, Exactive Plus, and QExactive are trademarks and Exactive, Orbitrap, Orbitrap Fusion, and Xcalibur are registered trademarks of Thermo Fisher Scientific Inc. KEGG is a registered trademark of Minoru Kanehisa (an individual). ChemSpider is a registered trademark of ChemZoo Inc.

mzCloud is a registered trademark of HighChem, Ltd. in the Slovak Republic.

Microsoft and Excel are registered trademarks of Microsoft Corporation in the United States and other countries.

All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.

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