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To familiarize yourself with the Mass Frontier™ 8.0 SR1 application, follow the topics in this tutorial to detect the
components in a chromatogram, identify the components using an mzCloud™ library search, annotate one of the
components, and save its annotated spectral tree to a local user library.
Contents
• Prerequisites
• Demo Data Files
• Creating a User Library
• Opening a Raw Data File and Detecting Components
• Filtering the Components List
• Identifying Components with an mzCloud Library Search
• Annotating a Component
• Sending an Annotated Component to the Data Manager
• Saving an Annotated Component to a Target Library
Prerequisites To follow the procedures in this tutorial, your processing computer must have installations of the Mass Frontier 8.0
SR1 and Server Manager 3.0 applications and an Internet connection with unblocked access to the mzCloud server.
Demo Data
Files
This tutorial uses the following file that is located in the Demo Data folder on the application computer.
To substitute the demo files with your own data, you must have an Xcalibur™ RAW file from an LC/MS/MS
experiment.
Creating a
User Library
Use the Server Manager 3.0 application to create user libraries for your mass spectral data.
To create a user library
1. To open the Server Manager, do one of the following:
• From the Windows™ 7 Start menu, choose (All) Programs > Thermo Mass Frontier 8.0 > Mass Frontier
Server Manager 3.0.
• From the Windows 10 Start menu, choose Thermo Mass Frontier 8.0 > Mass Frontier Server Manager
3.0.
• From the desktop, double-click the Mass Frontier Server 3.0 icon, .
Saving Compounds to Mass Frontier Spectral Libraries
Tip To check the connection to the mzCloud server, from the Mass Frontier application tab bar, click Start to
open the Start menu, and then choose Connection Check. In the Connection Check dialog box, click Run.
File Description
Sophora_P_Inj011MS.raw LC/MS/MS analysis of an extract from a sophora japonica flower acquired
on a Thermo Scientific™ Orbitrap Fusion Lumos™ MS platform. The
flowers of the sophora japonica plant have medicinal properties.
Revision A XCALI-98167
2
Figure 1. Server Manager 3.0 window
2. In the Actions pane, of the Start Page, click Create Library.
The Create Library page opens. You must enter the database name in the Name box; the display name is
optional.
3. For this tutorial, do the following:
a. In the Name box, type Flavonoids.
The Create Library tab changes to the Create Database - Database Name tab.
b. In the Display Name box, type My Flavonoids.
4. Click Create.
5. Close the Server Manager application.
Note The database name and the display name can contain only alphabetical and numeric
characters and cannot be more than 123 or 128 characters in length, respectively.
Note After you complete this tutorial, you can delete the new user library.
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Opening a
Raw Data File
and Detecting
Components
Use the Chromatogram Processor module to open the raw data file and detect components.
Follow these topics in the order listed:
1. Opening a Raw Data File
2. Detecting Components in an LC/MS Chromatogram
Opening a Raw
Data File
To open the example raw data file
1. Open the Mass Frontier application by double-clicking its desktop icon, , or by choosing Thermo Mass
Frontier 8.0 > Mass Frontier 8.0 from the Windows™ Start menu.
The application opens to the Mass Frontier startup window or the Modules & Tools toolbar.
2. Do one of the following:
• From the Mass Frontier startup window, under Open, click Chromatogram Processor.
Figure 2. Mass Frontier startup window
–or–
• In the Modules & Tools toolbar, click Chromatogram Processor.
3. In the Open Chromatogram dialog box, browse to the following folder, select Sophora_P_Inj011MS.raw, and
click Open.
drive:\Users\Public\Public Documents\HighChem\Mass Frontier 8.0\Demo Data\Chromatograms
Note If you clear the Show this Window Next Time check box, the next time you open the application,
it opens with the Modules & Tools toolbar displayed.
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A new instance of the Chromatogram Processor module opens as a tabbed document with the following views in
clockwise order (Figure 3):
• The chromatogram data view at the upper left lists the scan data by scan stage and number.
• The chromatogram view at the upper right displays the total ion current (TIC) chromatogram. The y-axis
scale is set to absolute intensity.
• The MS spectrum view at the lower right displays the first scan in the raw data file.
• The command processor view at the lower left is empty until you apply actions to the chromatogram.
Figure 3. TIC chromatogram and scan number 1 for the selected raw data file
Note Applying a component detection algorithm to the chromatogram adds a list of detected
components to this view.
Note To change the scale from absolute counts to relative intensity (versus the base mass spectrum peak),
right-click the view and choose Show Absolute Intensities.
Note Large data files can take a significant time to load. The status bar at the bottom of the application
window provides information about the loading progress, from reading the scan data to building the scan
tree
Tip To show or hide the views on a Chromatogram Processor page, click the following icons in the View
group of the Chromatogram Processor toolbar:
• For the MS spectrum view, click the Show MS Spectrum icon, .
• For the chromatogram data view, click the Show Chromatogram Data icon, .
• For the command processor view, click the Show Command Processor icon, .
You cannot hide the chromatogram view.
Command
processor view
Chromatogram data
view
Spectrum page of
the Spectrum view
TIC page of the
chromatogram view
5
Detecting
Components in
an LC/MS
Chromatogram
Use the Joint Component Detection (JCD) algorithm to detect the components in the example data file from an
LC/MS experiment.
To detect the components in a chromatogram from an LC/MS experiment
1. In the Actions group of the Chromatogram Processor toolbar, click JCD.
Figure 4. Actions group
The Joint Component Detection view opens on the right.
2. To make sure that you are starting with the default parameter settings, click the Reset Parameters to the Default
State icon .
3. For Tree Branching, select the Each Parent Ion Produces an Individual Tree option.
Figure 5. Joint Components Detection view
4. To detect components and create a spectral tree, click Preview.
Note For details about the component detection algorithms, refer to the Chromatogram Processor Module chapter
in the Mass Frontier 8.0 SR1 User Guide.
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After processing finishes, the following items appear:
• In the chromatogram data view, the Components list appears above the MS1 Scans list. The components are
displayed in chromatographic order (ascending order by retention time). The number of detected
components appears at the bottom of the view.
Figure 6. Components list
• On the TIC page of the chromatogram view, blue triangles appear above the apexes of the chromatographic
peaks for the detected components.
• On the left of the MS spectrum view, a spectrum tree depicts the selected component as a hierarchically
arranged set of nodes with different ion activation types and collision energies and stacks of replicate scans
grouped by an identical precursor pathway.
5. Click Accept.
The JCD view closes and the applied action appears in the command processor view.
Filtering the
Components
List
Running an mzCloud search requires fragmentation data. To display only the components that have associated
fragmentation scans, filter the components list in the chromatogram data view by the MSn stage.
To filter the components list by the MSn stage
In the MSn column of the chromatogram data view, click the filter icon, , and select 2.
The MS2 filter reduces the number of displayed components to 227 (Figure 7 on page 7).
Tip You can change the color-coding for the ion activation types. To change the color-coding, open the
MS Tree view of the Options dialog box by clicking Start > Global Settings > MS Tree.
Note The Sophora_P_Inj011MS.raw file contains only MS1 and MS2 data. If the raw data includes more scan
stages, use the Filter Editor to set the filter to Stage 2.
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Figure 7. Filtered Components list
Identifying
Components
with an
mzCloud
Library
Search
To identify the components in the example chromatogram, run an mzCloud Identity search.
To identify the components in the example chromatogram
1. In the Search group of the Chromatogram Processor toolbar, click Components Search.
The Component Search view opens to the right of the chromatogram and MS spectrum views.
2. In the Search Type list, select Identity.
3. In the Library list, select the Reference check box under mzCloud libraries.
Figure 8. Component Search view
4. To search the mzCloud mass spectral library for matching compounds for all the components, click Search All.
When the search ends, you can check for identified components in the chromatogram data view. For each
identified component in the Components list, the name and confidence score for the best matching library entry
appears in the Match column. The Annotation Sources column lists the search type.
Note An identity search looks for library entries where the m/z value of the precursor ion matches that of the
selected component (within the specified mass tolerance). It then compares the MS2 query spectra to the
MS2 library spectra.
Match score and name of the matching
compound in the mzCloud library
Search type
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5. In the Components list, select Component 2.
6. In the Component Search view, a list of matching entries appears for component 2 (Figure ).
Matching mzCloud library entries for component 2
Annotating a
Component
An mzCloud Identity search uses MS2 fragmentation data to confidently identify an unknown component, and a
higher score increases the confidence.
To annotate a component with the name and structure of the mzCloud hit
1. To sort the Components list by the match score in descending order, click the Match column until its arrow
points down, or right-click the Match column and choose Sort Descending (Figure 9).
Note You do not need to select a component in the Components list before running a library search on all
the components. However, if no component is selected, the following instruction appears in the Component
Search view—Please select a component. If the search finds no matching spectra for the selected
components, the following message appears—No results for Component X in mzCloud Reference Using
Identity.
Tip There are two ways to sort the columns in the Components list.
• To sort a single column, click the column heading until the desired arrow appears in the
column ( for descending, or for ascending), or right-click the column heading and choose
a sort order.
The arrow to the right of a column heading indicates that the column is sorted. When you
clear the sorting for a column, the arrow disappears.
–or–
• To sort by multiple columns:
a. Sort the first column.
b. Press the SHIFT key then click on the next column to sort. Repeat this step to sort by
another column.
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Figure 9. Components list sorted by match score
2. In the Components list, select Component 102 (taxifolin).
3. To assign the proposed structure to the selected component, click the Accept Structure icon, , in the
Component Search view.
In the chromatogram data view, the annotated component moves to the top of the Components list, and its
name and confidence score appear in bold font. Accepting a structure also links the structure to the component.
Note Taxifolin is a plant flavonoid.
Tip To annotate multiple components, select them in the Components list, and then click the Accept All
Suggested Structures icon, , in the Component Search view.
Sorted in descending order by the match score
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Sending an
Annotated
Component to
the Data
Manager
To send the annotated component to the Data Manager
1. In the Components list, select the annotated component (component 102 annotated as Taxifolin).
2. In the Send To group of the Chromatogram Processor toolbar, click Component and select New Data Manager.
A new instance of the Data Manager module opens as a tabbed page with a new spectral tree on the left, a
spectrum in the middle, and the assigned structure for the selected component on the right.
Saving an
Annotated
Component to
a Target
Library
After you send an annotated component to the Data Manager module, save the component to the appropriate user
library (target library).
To save the annotated component to a user library
1. In Library group of the Data Manager toolbar, click Save Changes.
2. In the Save Changes dialog box, select the target library.
3. Click OK.
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4. Click Save.
The application saves the entry to the target library and clears the page.
5. To view the compound in the target library, do the following:
a. On the Data Manager page, click the Target Library tab.
b. Select the entry that you want to view (Figure 10).
Figure 10. Data Manager showing the entry for taxifolin in the My Flavonoids library
Trademarks Mass Frontier, mzCloud, and Spectral Tree are trademarks of Thermo Fisher Scientific; and Xcalibur and Orbitrap
Fusion Lumos are a registered trademarks of Thermo Fisher Scientific Inc. in the United States.
Windows is a registered trademark of Microsoft Corporation in the United States and other countries.
All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.
Target library tab
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