Mass Frontier 7.0 Quick Start Guide
The topics in this guide briefly step you through key features of the
Mass Frontier™ application.
• Editing a Structure
• Working with Spectral Trees
• Building a Library
• Predicting Fragmentation and Automatically Annotating Spectra
• Performing Component Detection
• Matching Components to Library Entries
• Performing a Fragment Ion Search (FISh)
The procedures use the buspirone data files that are included with the
Mass Frontier application.
Note The Mass Frontier application supports data acquired
through the Thermo Xcalibur data system, version 1.4 or later.
Editing a Structure
You can import, edit, check, or export chemical structures in the
Mass Frontier application using the Structure Editor module, which
supports structure files with the .mol, .sdf, or .mcs file extension.
Y To open a structure
1. In the Mass Frontier main window, do one of the following:
Choose File > Open > Structure.
– or –
Click the Structure Editor button,
, in the toolbar
and then click the Insert Structure button,
, in the
Structure Editor dialog box.
2. In the Open Structure dialog box, select the buspirone.mol file
from this folder:
…\Documents and Settings\All Users\Shared Documents
\HighChem\Mass Frontier 7.0\Structures
3. Click Open.
4. In the Structure Editor dialog box, experiment with some of the
drawing tools as follows:
Click a symbol,
and then click
the workspace to
add the symbol.
Revision A XCALI-97350
© 2011 Thermo Fisher Scientific Inc.
All rights reserved.
Working with Spectral Trees
The Mass Frontier application uses a tree representation of the data
structure of MSn spectra. A spectral tree, which is like a structural
“fingerprint,” reflects the hierarchical spectra dependencies in MSn
experiments.
A spectral tree consists of the following:
• Levels—MSn stages starting at n=1
• Node connectors—Graphical symbols of the precursor m/z value,
or the precursor m/z range if the isolation width is included
• Node items—Product or calculated spectra of the identical
precursor m/z value or m/z range, or for a chromatogram
• Nodes—Containers of the node items
Spectral tree example
MS1
MS/MS
3
MS
Parallel product spectra for a given node can be
from any precursor ion, including spectra acquired
at various collision energies and isolation widths
or any other spectra that enhance reproducibility
in compound identification.
You can store spectral trees in the libraries that you create using the
Mass Frontier application. You can also associate any complementary
information connected with a single-stage spectrum or a
chromatogram with a node spectrum or node chromatogram.
Methods for Creating Spectral Trees
Use any of the following methods to create a spectral tree:
• Perform component detection and spectral deconvolution on a
Data Dependent™ MSn chromatogram.
• Manually create the spectral tree.
• Create a spectral tree from various MSn experiments (spectra) in a
single sample file (direct infusion) or from multiple sample files
stored in a single folder. (The procedure for this method follows.)
For detailed procedures about using these methods, refer to the Help
within the Mass Frontier application or the Mass Frontier User Guide.
Click to undo
any unwanted
change.
Click to open
the Templates
dialog box and
view the template
options.
5. Close the Structure Editor dialog box without saving your
changes. You will use the original buspirone.mol file in a
later step.
Click to view the options in
the Unspecified Charge list.
Creating a Spectral Tree from Multiple MSn Spectra in
a Single Direct-Infusion Sample File
The component symbol turns green,
and the spectral tree appears.
Before you start this procedure, locate the
directinfusionbuspirone.raw file:
…\Documents and Settings\All Users\Shared Documents\
HighChem\Mass Frontier 7.0\Chromatograms\Direct Infusion
Or, you can download the file from the HighChem Web site at
www.highchem.com/directinfusionbuspirone.raw.
Y To create a spectral tree from a sample file
1. Choose Tools > Chromatogram Processor, or click the
Chromatogram Processor button,
, in the Mass Frontier
toolbar.
2. In the Open Chromatogram dialog box, select the
directinfusionbuspirone.raw file, and then click Open.
3. In the Chromatogram Processor window, perform component
detection on the direct infusion sample file as follows:
Now you are ready to add the component 1 spectral tree to a library.
a. Click the Components Detection
& Spectra Deconvolution button,
and then choose Direct Infusion.
Building a Library
Use the Database Manager module to create and organize spectral
and chromatographic libraries with chemical structures. Because the
Mass Frontier application supports ion structures and the tree spectra
representation, you can create true MSn libraries.
Follow these steps to copy the spectral tree to the database, create a
library, and then add the spectral tree to your library.
Step 1. Create a Library
1. Choose Library > Server Manager in the Mass Frontier main
window.
2. In the Mass Frontier Server Manager window, create a library as
shown in the following steps:
a. Right-click the server name, and then
choose Create Library.
b. Click Default to reset the values as shown.
c. Click Calculate.
d. After the spectral tree
construction detection is
finished, click Close.
b. Type a name in the
Library Name box.
Tip To zoom in on a chromatogram, drag the cursor to form a
box around the area to be enlarged.
4. In the TIC pane, click the triangle that indicates a component.
The component symbol turns green, and the spectral tree of
the component appears in the Spectrum pane. In the list,
component 1 (m/z 386.30) is highlighted.
2
c. Click
Create.
3. After creating the library, close the Mass Frontier Server Manager.
4. To make the new library accessible to the application, close the
Mass Frontier application and restart it.
Predicting Fragmentation and
Automatically Annotating Spectra
The Mass Frontier Fragmentation Library™ uses 24 general
ionization, fragmentation, and rearrangement rules and
approximately 31 000 fragmentation schemes collated from
published literature to automatically predict fragmentation,
rearrangement, and resonance mechanisms from a chemical
structure. The structure can include ions and isotopically labeled
compounds.
You can start the fragmentation process from the Structure Editor
window, the Database Manager window, or the Fragments &
Mechanisms window. When you start the process from the Database
Manager window, the Mass Frontier application automatically links
the generated fragments in the Fragments & Mechanisms window
with the corresponding spectrum in the Database Manager window.
In the spectrum, peaks that have the same m/z values as the generated
fragments appear in red, different from the original spectrum in blue.
You can select a red peak to see all the pathways leading to the peak.
The following procedure shows how to predict the fragmentation
pathways for the buspirone structure, compare the theoretical
fragmentation to the data from the experimental direct-infusion
sample, and then automatically annotate a spectrum.
Tip To remove a user library, go to the Mass Frontier Server
Manager, right-click the user library and select Uninstall Library.
Step 2. Copy the Spectral Tree to the Database
1. In the Chromatogram Processor window, select component 1.
2. In the Mass Frontier main toolbar, click the Copy button,
,
and select Copy MS Tree.
3. In the Mass Frontier main toolbar, click the Database Manager
button,
. A new Database Manager window opens.
4. In the Mass Frontier main toolbar, click the Paste button,
,
and choose Tree.
Step 1. Generate Possible Fragmentation and
Rearrangement Pathways
1. Make sure the Database Manager window that contains the
spectral tree from the direct-infusion sample is still open, and
then open the buspirone.mol structure as shown in the next
figures.
Tip In the Database Manager toolbar, click the Show Spectral
Tree button,
, to switch the spectral tree pane in the window.
Step 3. Add the Record to Your Library
1. In the Mass Frontier main window, select the Database Manager
window and choose Library > Add To Library from the
Mass Frontier main menu.
2. In the Add To Library dialog box, do the following:
a. Click the Open button in the Mass Frontier toolbar, and then choose Structure.
Select the library you
created, and then
click Add.
In the Database Manager
window, the new entry appears
in the New Demo tab.
b. Select buspirone.mol and click Open. The file is in the following folder:
… \Documents and Settings\All Users\Shared Documents \HighChem
\Mass Frontier 7.0\Structures
Leave the Database Manager window open for the next procedure,
which compares the theoretical fragmentation of a buspirone
structure to the experimental direct-infusion buspirone sample.
3
4. In the Fragments & Mechanisms window, view the results:
The application displays the molecular structure in the structure
pane and the molecular mass and formula on the Work page.
a. Click the Fragments tab to show only the Fragments,
or click the Mechanisms tab to show both
mechanisms and fragments.
Tip In the Database Manager toolbar, click the Show
Structure button,
, to switch the structure pane in the
window.
2. In the Mass Frontier main window, with the Database Manager
window selected, do the following:
Click the Fragments and Mechanisms button.
b. Click an m/z tab or value to display the
results for that value.
Step 2. Compare the Theoretical Fragments to the
Experimental Data
1. In the Database Manager window, do the following:
a. Click a node of the spectral tree to
display the corresponding spectrum.
Molecular mass and formula
b. In the spectrum, click a red peak.
Molecular structure
3. In the Reaction Restrictions dialog box, do the following:
a. Click Restore Defaults.
b. Select the
Both option.
c. Select the
Active check
boxes for all
libraries.
The Fragments & Mechanisms window displays the possible
fragments and mechanisms for the selected red peak.
d. Click OK.
The Generation of Fragments & Mechanisms window opens.
In the spectrum, the red peaks are “explained” peaks that can be
linked to the generated fragments from the buspirone structure
based on m/z values. The blue peaks in the spectrum cannot be
linked to the generated fragments.
The Mass Frontier application generates all the m/z values for the
given reaction restrictions settings and displays the results in the
Fragments & Mechanisms window.
4
2. In the Fragments & Mechanisms window, change which
m/z values appear as follows:
Performing Component Detection
a. Click the Show m/z Values for Explained Peaks Only button.
Only the fragments for m/z values that can be linked with a spectrum
(explained peaks) appear.
b. Click the button again to display results for all the m/z values.
You can choose from several algorithms when performing component
detection on data from a Data Dependent MSn experiment. Thermo
Fisher Scientific recommends the JCD algorithm for most of your
LC/MS experiments.
Y To perform component detection
1. In the Mass Frontier toolbar, click the Chromatogram Processor
button,
.
2. In the Open Chromatogram dialog box, select the buspirone.raw
file in the following folder and click Open.
…\Documents and Settings\All Users\Shared Documents
\HighChem\Mass Frontier 7.0\Chromatograms
3. In the Chromatogram Processor toolbar, click the Components
Detection & Spectra Deconvolution button,
, and
choose JCD.
4. In the J-Component Detection Algorithm dialog box, set these
parameters:
Step 3. Automatically Annotate the Spectra
1. In the Database Manager window, right-click the spectrum and
choose Auto Annotation > Generated Fragments.
c. Click Calculate.
d. Click Close.
a. Click Default to
reset the parameter
values.
b. Change the MS Stage
to 2.
2. In the Threshold box, type 5.00, select the Apply to All Nodes
check box to complete annotation for the entire tree, and click
OK.
5. In the TIC pane, view the detected components. You can click on
each
to view the selected ion profile and the corresponding
ion tree.
Current active
component
3. When prompted with the number of new fragments found in the
generated fragments and mechanisms, click OK.
4. In the Spectrum pane, right-click and choose Auto Annotation
Layout. When prompted to rearrange the annotations for all tree
nodes, click Yes.
Now you can clearly view each annotation.
List of detected
components
5
Ion tree of the
active component
MSn spectrum of
the active node
5. Click the Hit Selector button,
, to view the matched library
entry in the Hit Selector window.
Matching Components to Library Entries
You can match the components detected in the buspirone.raw file
against the library entries.
Y To match components to library entries
1. In the Chromatogram Processor window TIC pane, zoom in on
the components between retention time 10.00 and 12.00.
6. Click OK to accept the assignment.
2. In the Chromatogram Processor toolbar, click the
Components Editor button,
. The information for the
selected components appears in the Components Editor window.
3. In the Components Editor window, start the component search
as follows:
Performing a Fragment Ion Search (FISh)
You can use Fragment Ion Search (FISh) to quickly screen for
structure-related compounds (compounds with common fragments).
Y To perform a fragment ion search
1. Generate fragment ions for the compound of interest (see
“Predicting Fragmentation and Automatically Annotating
Spectra” on page 3).
2. In the Fragments and Mechanisms window, copy the list of
fragments as follows:
a. Click the Search button and
choose Search All Components.
a. In the Mass
Frontier toolbar,
click the Copy
button and
choose List of
Fragments.
b. In the
Chromatogram
Processor
window, click
the FISh
button.
b. Select the library you
created and click OK.
4. On the Work page in the Components Editor window, use the
vertical scroll bar to view the component list and look for
matches.
The application assigns a score
(match factor) to all matches.
Heat Selector
button
c. In the FISh Filter
dialog box, click
Default, and
then click the
Edit button
under
Fragments
(empty).
Continue to next figure.
6
d. In the FISh Filter: Input
Fragments dialog box,
click the Paste button
to paste the copied
fragments from the
Clipboard, and then
click OK.
Useful Window Icons
The Mass Frontier 7.0 application consists of more than a dozen
frequently used windows. The next table shows the icons of these
windows and their corresponding definitions.
Icon
e. Click
Calculate,
and when the
calculation is
done, click
Close.
3. In the Chromatogram Processor window, select the visualization
options.
a. Click the Visualization Options pin.
b. Clear the Show TIC and Show All
Components check boxes.
Definition
Icon
Definition
Structure Editor
Formula Generator
Database Manager
Fragments and
Mechanisms
Chromatogram
Processor
Report Creator
Spectra Classifier
Chromatogram
Processor Utilities
Fragments
Comparator
Components
Detection & Spectra
Deconvolution
Isotope Pattern
Components Editor
Periodic Table
Getting Help
For additional information, consult the following:
• For more details about using the Mass Frontier application, refer
to the videos and the Help available from the Mass Frontier Help
menu or the Mass Frontier User Guide.
• For the latest service release of the software, visit
www.highchem.com/downloads/mass-frontier.
• For upcoming application notes and posters, visit
www.thermo.com.
• For Technical Support:
Phone: 1-800-532-4752
Fax: 1-561-688-8736
E-mail: [email protected]
For international contacts, go to
http://www.thermo.com/cda/contactus/home
4. In the Chromatogram Processor window, view the FISh result.
Pink peaks represent matched ions.
View the filtered chromatogram by FISh in pink.
Mass Frontier and Fragmentation Library are trademarks of
HighChem, Ltd.
Data Dependent is a trademark and Xcalibur is a registered
trademark of Thermo Fisher Scientific Inc.
An MS/MS spectrum of a buspirone hydroxylated metabolite is
displayed. Matched fragments appear in pink.
You can combine the FISh results with other functions in the
application to perform further data manipulation. For example,
locate the corresponding components based on the retention time
and precursor information and add them to a user-created library.
7