Mass Frontier 7.0 Quick Start Guide

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1 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. 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 to undo any unwanted change. Click a symbol, and then click the workspace to add the symbol. 5. Close the Structure Editor dialog box without saving your changes. You will use the original buspirone.mol file in a later step. Working with Spectral Trees The Mass Frontier application uses a tree representation of the data structure of MS n spectra. A spectral tree, which is like a structural fingerprint, reflects the hierarchical spectra dependencies in MS n experiments. A spectral tree consists of the following: Levels MS n 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 MS 1 MS/MS MS 3 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 MS n chromatogram. Manually create the spectral tree. Create a spectral tree from various MS n 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 open the Templates dialog box and view the template options. Click to view the options in the Unspecified Charge list. Revision A XCALI Thermo Fisher Scientific Inc. All rights reserved.

2 Creating a Spectral Tree from Multiple MS n Spectra in a Single Direct-Infusion Sample File 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 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: a. Click the Components Detection & Spectra Deconvolution button, and then choose Direct Infusion. Now you are ready to add the component 1 spectral tree to a library. Building a Library The component symbol turns green, and the spectral tree appears. 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 MS n 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. 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 ) is highlighted. b. Type a name in the Library Name box. c. Click Create. 2

3 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. 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 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. Tip In the Database Manager toolbar, click the Show Spectral Tree button,, to switch the spectral tree pane in the window. Predicting Fragmentation and Automatically Annotating Spectra The Mass Frontier Fragmentation Library uses 24 general ionization, fragmentation, and rearrangement rules and approximately 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. 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. 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. 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. 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 3

4 The application displays the molecular structure in the structure pane and the molecular mass and formula on the Work page. Tip In the Database Manager toolbar, click the Show Structure button,, to switch the structure pane in the window. 4. In the Fragments & Mechanisms window, view the results: a. Click the Fragments tab to show only the Fragments, or click the Mechanisms tab to show both mechanisms and fragments. 2. In the Mass Frontier main window, with the Database Manager window selected, do the following: Click the Fragments and Mechanisms button. Molecular mass and formula Molecular structure 3. In the Reaction Restrictions dialog box, do the following: a. Click Restore Defaults. 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. b. In the spectrum, click a red peak. b. Select the Both option. c. Select the Active check boxes for all libraries. d. Click OK. The Fragments & Mechanisms window displays the possible fragments and mechanisms for the selected red peak. 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

5 2. In the Fragments & Mechanisms window, change which m/z values appear as follows: 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. Step 3. Automatically Annotate the Spectra 1. In the Database Manager window, right-click the spectrum and choose Auto Annotation > Generated Fragments. Performing Component Detection You can choose from several algorithms when performing component detection on data from a Data Dependent MS n experiment. Thermo Fisher Scientific recommends the JCD algorithm for most of your LC/MS experiments. 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: c. Click Calculate. d. Click Close. a. Click Default to reset the parameter values. 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. b. Change the MS Stage to 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 Ion tree of the active component MS n spectrum of the active node 5

6 Matching Components to Library Entries You can match the components detected in the buspirone.raw file against the library entries. To match components to library entries 1. In the Chromatogram Processor window TIC pane, zoom in on the components between retention time and Click the Hit Selector button,, to view the matched library entry in the Hit Selector window. 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: a. Click the Search button and choose Search All Components. 6. Click OK to accept the assignment. Performing a Fragment Ion Search (FISh) You can use Fragment Ion Search (FISh) to quickly screen for structure-related compounds (compounds with common fragments). 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. 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 Continue to next figure. c. In the FISh Filter dialog box, click Default, and then click the Edit button under Fragments (empty). 6

7 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 Definition Icon Definition Structure Editor Formula Generator 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. Database Manager Chromatogram Processor Spectra Classifier Fragments Comparator Isotope Pattern Periodic Table Fragments and Mechanisms Report Creator Chromatogram Processor Utilities Components Detection & Spectra Deconvolution Components Editor 4. In the Chromatogram Processor window, view the FISh result. Pink peaks represent matched ions. View the filtered chromatogram by FISh in pink. 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 For upcoming application notes and posters, visit For Technical Support: Phone: Fax: For international contacts, go to 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

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