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质谱图 Spectrum: Getting Started To Display the First Scan of the Current Data File To Display a Scan at a Particular Time in the Current Data File To Display the First Scan of the Current Data File Select the MassLynx Sample List Menu Bar Spectrum command, the first scan of the current data file is displayed. To Display a Scan at a Particular Time in the Current Data File Either: Double-click at the required time (on the X-axis) in the Chromatogram display. The Spectrum for that time is displayed. Or: Select the Spectrum Tool Bar button. The Select Raw Spectrum dialog is invoked; the Entry: box displays the time for the currently-displayed spectrum. a. Enter the time for the required Spectrum in the Entry: text box. b. Select the OK button. The Spectrum for that time is displayed. Or: Select the Spectrum Menu Bar Display, Spectrum, At command. The Display Raw Spectrum dialog is invoked; the Spectrum: text box displays the time for the currently displayed spectrum. a. Enter the time for the required Spectrum in the Spectrum: text box. b. Select the OK button. The Spectrum for that time is displayed. The Spectrum Window The Spectrum module runs in a top-level window that has a Menu Bar and Tool Bar at the top. The top-level window may contain one or more Spectrum Windows; each can contain one or more Spectrum traces. When there is more than one trace in a window, the current trace is identified by a colored square at the left of the trace. To select another trace, click on any part of the trace, or select a trace from the Menu Bar Display, Graphs command, or use the keyboard up and down arrow keys. The spectra in each Spectrum window share a common mass axis; place Spectra in separate windows to display them on different mass axes. The Spectrum Menu Bar The Spectrum File Menu The Spectrum Edit Menu The Spectrum Display Menu The Spectrum Process Menu The Spectrum Tools Menu The Spectrum Window Menu The Spectrum Help Menu The Spectrum File Menu Open Opens a data file. Save Spectrum Saves a processed spectrum, see To Save a Processed Spectrum. Note: This option is only available when a processed spectrum is selected. Export Allows the export of data to an ASCII file consisting of spectrum masses and intensities, see Exporting SEQUEST Files. SEQUEST file Note: This option is only available for BioLynx and non-GC installations. Print Prints the current Spectrum Window. Print Report Prints a list of spectrum masses and intensities, see To Print a Report of the Spectrum Listing. Invokes the standard Windows Print Setup dialog. Printer Setup Exit Closes the Chromatogram window. The Spectrum Edit Menu Copy Picture Copies the current Window to the clipboard. Copy Spectrum Copies the currently-displayed range of the spectrum trace to the clipboard as mass and intensity pairs in the form of a text list. List Paste Pastes the clipboard contents into the display. Invokes the standard Windows Paste Special dialog. Paste Special Library Invokes the Library sub-menu, used to append spectra to user libraries and to view spectra in any library. Invokes the Display Library Spectrum dialog, which allows a Get Library entry to be displayed, see To Display a Library Entry. Spectrum Invokes the Append Spectrum dialog, which is used to append the Append current spectrum to the Library, see To Append the Current Spectrum to the Current Library. The Spectrum Display Menu Spectrum Invokes the Spectrum sub-menu, see The Spectrum Display, Spectrum Sub-Menu. Invokes the Remove Spectra dialog, used to remove multiple Remove spectrum traces from the display; see To Remove Multiple Spectrum Traces from the Display. Invokes the Spectrum Real-Time Update dialog, used to display Real-Time new spectra as they are being acquired, see Real-Time Display of Update Spectra Range Invokes the Range sub-menu, see The Spectrum Display, Range Sub-Menu. List Spectrum Displays a spectrum as a list of peak masses and intensities, see Displaying a Spectrum as a List. Invokes the Spectrum Display dialog, used to change the View spectrum display parameters, see Controlling the Appearance of the Display. Invokes the Spectrum Peak Annotation dialog, used to edit the Peak Peak Annotation Parameters; see Controlling the Appearance of Annotation Peak Labels. Invokes the Customize Toolbar dialog; see Customizing the Customize Spectrum Tool Bar. Toolbar Toolbar Toggles the Tool Bar on and off. Status bar Toggles the Status Bar on and off. Move to Last Moves the currently selected Spectrum to the top of the display, see Changing the Order of Displayed Spectra. Move to First Moves the currently selected Spectrum to the bottom of the display, see Changing the Order of Displayed Spectra. Graphs Displays a list of the Spectra in the display; click on a Spectrum in the list to select it. More: The Spectrum Display, Spectrum Sub-Menu The Spectrum Display, Range Sub-Menu The Spectrum Display, Spectrum Sub-Menu Invokes the Display Raw Spectrum dialog, this allows the User to At view a spectrum, see To Display a Scan at a Particular Time in the Current Data File. Invokes the Display Quan DB Spectrum dialog, this allows the Peak List User to view a Peak List entry, see Viewing a Peak List Entry. Entry The Spectrum Display, Range Sub-Menu Invokes the Display Range dialog, used to change the mass axis From range; see To Alter the Range of the Mass Axis using the Menu Bar. Invokes the Spectrum Magnify dialog, used to magnify a section of Magnify the current spectrum trace; see Setting Magnification Ranges using the Menu Bar Magnify Command. Invokes the Default Spectrum Range dialog, to specify the default Default mass axis range; see To Change the Default Display Range. The Spectrum Process Menu Note: Spectrum only enables those processes that can be applied to the currently loaded data; hence, menu commands that are not applicable to the data are grayed-out. Invokes the Refine Spectrum dialog, used to automatically remove Refine background ions from a spectrum thereby allowing it to be more easily identified, for example by library search, see The Refine Process. Note: The Refine process operates on centroid-mode data only. Invokes the Combine Spectrum dialog, used to produce a single Combine spectrum by subtracting averaged background spectra from the average of spectra from a TIC peak, see The Combine Spectra Process. Invokes the Background Subtract dialog, used to adjust the zero Subtract level in a continuum spectrum to lessen the effect of chemical noise caused by column bleed, etc, see The Background Subtract Process. Invokes the Spectrum Smooth dialog, used to reduce the Smooth high-frequency noise present in a spectrum, thus aiding interpretation, see The Smooth Process. Invokes the Spectrum Center dialog, used to calculate the mass of Center the peak center, see The Center Process. Note: This option is disabled for non-continuum data. Invokes the Mass Measure dialog, used to center peaks with Mass Measure optional background subtraction and/or smoothing, on continuum spectra, see The Mass Measure Process. Select once to process all traces in the current window. Select Process All again to process only the current trace in the current window. Traces Component Invokes the Component sub-menu, see The Spectrum Process, Component Sub-Menu. Note: This Component sub-menu is only available for ElectroSpray data. ElectroSpray data consists of a series of multiply-charged ions. This series identifies a component that is used in the Transform and MaxEnt processes. Invokes the Transform dialog, see To Transform an ElectroSpray Transform Spectrum onto a Molecular Mass Axis; The Transform dialog. Invokes the MaxEnt 1 dialog, used to produce true molecular mass MaxEnt 1 spectra from multiply-charged ElectroSpray spectra, see MaxEnt 1. MaxEnt Errors Calculates a probable error range for the mass of each peak in the MaxEnt spectrum, see MaxEnt Errors. Invokes the Set Adduct Mass dialog used to set the adduct mass Set Adduct for MaxEnt and Transform, see Setting Adduct Mass for Mass Transform and MaxEnt. Invokes the MaxEnt 2 dialog, used to increase resolution and MaxEnt 2 remove noise for any singly charged continuum spectrum, see MaxEnt 2. Invokes the MaxEnt 3 dialog, used to resolve the multiply-charged MaxEnt 3 peaks onto a singly-charged axis for any low mass, multiply-charged continuum spectrum, see MaxEnt 3. Invokes the Peak Detect dialog, used to locate spectral peaks, Integrate draw baselines and calculate peak areas, see To Integrate a Spectrum. Invokes the TOF Transform dialog, see The TOF Transform TOF Transform Process. More: The Spectrum Process, Component Sub-Menu The Spectrum Process, Component Sub-Menu Invokes the Edit Components dialog, used to edit the components Edit stored in the component table, see Editing Components for Transform. Invokes the Automatic Find Components dialog, used to Find Auto automatically find components when the mass range is known, see To Find Components when the Mass Range is Known; the Automatic Find Components Dialog. Invokes the Manual Find Components dialog, used to manually Find Manual find components when the mass range is unknown, see To Find Components using the Manual Method; the Manual Find Components Dialog. The Spectrum Tools Menu Invokes the Library Search List dialog, used to identify the current Library Search scan using the library search facility, see Selecting Which Libraries to Search. Note: This option is only enabled for centroid data. Invokes the Isotope modelling dialog, used to produce an isotope Isotope Model cluster abundance plot for a given formula, see The Isotope modelling Dialog. Invokes the EleComp Parameters dialog, used to search for the Elemental possible component element(s) of a selected peak, see The Composition EleComp Parameters Dialog. ACD Labs Invokes the Advanced Chemistry Development's ACD/Spec Manager software suite for structural elucidation, (if installed on the SpecManager PC). Invokes the Make new calibration dialog, used to make a Make Calibration calibration using a reference file, see Performing a Calibration. Invokes the Apply Calibration dialog, used to apply the calibration Apply previously made using the Make Calibration command, see To Calibration Apply a Calibration Invokes the Modify Calibration dialog, used to modify the Modify calibration of a data file, see To Modify a Calibration. Calibration Invokes the Lock Mass dialog; this allows the User to specify a Lock Mass mass that will be located in the spectrum and used to calculate an offset that can be applied to the rest of the spectrum. See Lock Mass. The Spectrum Window Menu Tile Displays the current windows in a tiled view. Cascade Displays the current windows in a cascaded view. Stack Displays the current windows in a stacked view. Arrange Icons Arranges the icons of minimized windows at the bottom of the Chromatogram Window. Invokes the New Spectrum dialog see The New Chromatogram New Trace Dialog. Click on the required trace to select it. List of current traces The Spectrum Help Menu The Help, Spectrum command invokes the Help function for Spectrum. The Spectrum Tool Bar The Spectrum Tool Bar is displayed at the top of the Spectrum Window. The default Spectrum Tool Bar contains the buttons listed below. It is also possible to customize the Tool Bar and add additional buttons for other Spectrum operations. Menu equivalent Tool Purpose Bar button File, Open Opens a data file. File, Print Prints the current window in portrait format. File, Print Prints the current window in landscape format. Edit, Copy Picture Copies the current window to the clipboard. Edit, Copy Spectrum Copies the currently-displayed range of the List spectrum trace to the clipboard as mass and intensity pairs in the form of a text list. Edit, Paste Pastes the contents of the clipboard into the display. Process, Refine Refines the current scan. The refine process identifies the masses that contribute to a particular peak in the TIC. Tools, Library Search Identifies the current scan using the library search facility. Process, Process All Select once to process all traces in the current Traces window. Select again to process only the current trace in the current window. Invokes the Edit Text String dialog; this allows text to be added to a spectrum. Selecting once causes each subsequent spectrum to appear in a new window, rather than being added to the current one. Selecting a second time cancels this mode. Selecting once causes each subsequent spectrum to replace the currently selected trace. Selecting a second time cancels this mode. Note: The button is grayed when the button is depressed. Display, Real-Time Toggles real time spectrum data update on and off. Update Display, Range, Increases the magnification of the current range. Magnify Display, Range, Decreases the magnification of the current range. Magnify Display, Range, Deletes the current magnification range. Magnify Display, Spectrum, Selects a new scan from the current data file. At Decrements the currently displayed scan. Increments the currently displayed scan. Select once to restore the previous display range; select again to use the default display range. Customizing the Spectrum Tool Bar The Spectrum Tool Bar can be customized to: , Add buttons for frequently used operations. , Remove buttons that are not required. , Change the order in which the Tool Bar buttons are displayed. The additional buttons that can be added to the default Spectrum Tool Bar are: Menu equivalent Tool Purpose Bar button Saves the spectrum. File, Save Spectrum Process, Smooth Invokes the Spectrum Smooth dialog. Process, Combine Invokes the Combine Spectrum dialog. Process, Subtract Invokes the Background Subtract dialog. Process, Center Invokes the Spectrum Center dialog. Process, Mass Invokes the Mass Measure dialog. Measure Process, Component, Invokes the Manual Find Components Find Manual dialog. Process, Component, Invokes the Automatic Find Components Find Auto dialog. Process, Transform Invokes the Transform dialog. Tools, ACD Labs Invokes the Advanced Chemistry SpecManager Development's ACD/Spec Manager software suite for structural elucidation, (if installed on the PC). Window, Tile Tiles the windows. Window, Cascade Cascades the windows. Window, Stack Stacks the windows. More: The Customize Toolbar dialog To Add Buttons to the Tool Bar The Customize Toolbar dialog To customize the Spectrum Tool Bar, select the Spectrum Menu Bar Display, Customize Toolbar command; the Customize Toolbar dialog is invoked. Available This list box contains all the available buttons that are not Buttons: list currently in the Tool Bar. A button can be selected by box clicking on it. The top entry in the box is Separator; it is never removed from the Available Buttons: list box. However, it can be added to the Toolbar Buttons list box to insert a separation gap between the buttons in the Tool Bar. Tool Bar This list box contains all the buttons that are currently in the Buttons: list toolbar. A button can be selected by clicking on it. The last box entry in this box is always Separator (dimmed); it cannot be removed from the list box, it allows buttons to be added to the end of the list. Add-> Moves the selected button from the Available Buttons: list box to the Tool Bar Buttons: list box. <-Remove Moves the selected button from the Tool Bar Buttons: list box to the Available Buttons: list box. Note: This button is grayed if no item is selected in the Tool Bar Buttons: list box. Close Exits the Customize Toolbar dialog. Reset Resets the Tool Bar to its default display. Moves the selected button one position up the list in the Toolbar Move Up Buttons: list box. Note: This button is grayed if no item is selected in the Tool Bar Buttons: list box, or if the top item in the list is selected. Moves the selected button one position down the list in the Toolbar Move Down Buttons: list box. Note: This button is grayed if no item is selected in the Tool Bar Buttons: list box, or if the bottom item in the list is selected. To Add Buttons to the Tool Bar 1. Select the Spectrum Menu Bar Display, Customize Toolbar command; the Customize Toolbar dialog is invoked. 2. Select the button to be added in the Available Buttons: list box. 3. Select the Tool Bar button before which the new button is to be added in the Toolbar Buttons: list box. 4. Select the Add button. The new button is added to the Toolbar Buttons: list box. 5. Repeat steps 2 to 4 to add further buttons to the Tool Bar. 6. Separators can be inserted between Tool Bar buttons to divide them into logical groups. To add a separator, repeat steps 2 to 4 selecting Separator in the Available Buttons: list box. 7. Select the Close button to exit and save the changes. More: To Remove Buttons from the Tool Bar To Change the Order in which Tool Bar Buttons are Displayed To Reset the Tool Bar to the Default Settings To Remove the Tool Bar from the Spectrum Display To Remove Buttons from the Tool Bar 1. Select the Spectrum Menu Bar Display, Customize Toolbar command; the Customize Toolbar dialog is invoked. 2. Select the button to be removed in the Toolbar Buttons: list box. 3. Select the Remove button. The button is removed from the Toolbar Buttons: list box. 4. Repeat steps 2 and 3 to remove further buttons from the Tool Bar. 5. Select the Close button to exit and save the changes. To Change the Order in which Tool Bar Buttons are Displayed 1. Select the Spectrum Menu Bar Display, Customize Toolbar command; the Customize Toolbar dialog is invoked. 2. Select the button to be moved in the Toolbar Buttons: list box. 3. Select the Move Up or Move Down buttons to move the Tool Bar button. 4. Repeat steps 2 and 3 as often as required. 5. Select the Close button to exit and save the changes. To Reset the Tool Bar to the Default Settings 1. Select the Spectrum Menu Bar Display, Customize Toolbar command; the Customize Toolbar dialog is invoked. 2. Select the Reset button. 3. Select the Close button to exit and save the changes. To Remove the Tool Bar from the Spectrum Display Select the Menu Bar Display, Toolbar command, the Tool Bar will be removed from the display. A tick mark appears next to this menu item when it has been selected. To re-display the Tool Bar, select the Menu Bar Display, Toolbar command again. Displaying Spectra Adding or Replacing Spectra The New Spectrum Dialog Viewing a Peak List Entry Adding or Replacing Spectra MassLynx provides a number of options for displaying new spectrum traces. New spectrum traces can be generated by , Opening a new file. , Processing spectra (subtract, smooth, center, etc.). , Selecting spectra by double-clicking on a chromatogram. To display each new spectrum trace in a new window, select the Tool Bar button. To cancel this mode and display new traces in the existing window select the Tool Bar button again. When a new trace is displayed in the existing window, it can be added to the traces currently displayed, or it can replace the current trace. Select the Tool Bar button once to cause each subsequent spectrum, or spectrum process, to replace the currently selected trace. Selecting the button a second time causes each subsequent spectrum, or spectrum process, to be added to the traces on display. Up to sixteen spectrum traces can be displayed in one window. Note: 1. The button is grayed when the button is depressed. 2. The manner in which spectra are added to the Spectrum Window can also be selected via the Menu Bar Window, New Trace command, refer to The New Chromatogram Dialog. The New Spectrum Dialog The New Spectrum dialog is used to select the manner in which spectra are added to the Spectrum Window; it is invoked by the Menu Bar Window, New Trace command. Add Trace Adds the spectrum to the current Spectrum Window. Replace Trace The spectrum replaces the currently selected spectrum in the Spectrum Window. New Window Displays the spectrum in a new Window. Viewing a Peak List Entry To view a Peak List entry, select the Menu Bar Display, Spectrum, Peak List Entry command, this invokes the Display Quan DB Spectrum dialog. File: Displays the current file. Entry: Enter the required entry number, this field will only accept integer values in the range 1 to the number of entries in the Peak List. File Invokes the standard windows file Open dialog. Add Trace Adds the spectrum to the current Spectrum Window. Replace Trace The spectrum replaces the currently selected spectrum in the Spectrum Window. New Window Displays the spectrum in a new Window. Manipulating the Display Altering the Range of the Mass Axis Altering the Range of the Intensity Axis Altering the Range of Both Axes Setting Magnified Ranges Deleting Magnification Ranges Restoring the Display Setting the Display Range Defaults Displaying a Spectrum as a List Altering the Range of the Mass Axis To Alter the Range of the Mass Axis using the Mouse To Alter the Range of the Mass Axis using the Menu Bar To Alter the Range of the Mass Axis using the Mouse Click and hold the left mouse button at one end of the region of interest and drag the cursor horizontally to the other end. As the cursor is dragged, a "rubber band" is stretched out to indicate the range selected; do not go beyond the bounds of the axis. When the mouse button is released, the selected range will be re-displayed to fill the current window. This operation can be repeated as often as required. To Alter the Range of the Mass Axis using the Menu Bar 1. Select the Menu Bar Display, Range, From command. The Display Range dialog is invoked. 2. Enter new From and To values for the mass axis. 3. Select the OK button. Altering the Range of the Intensity Axis Click and hold the left mouse button at one end of the region of interest and drag the cursor vertically to the other end. As the cursor is dragged, a "rubber band" is stretched out to indicate the range selected; do not go beyond the bounds of the axis. When the mouse button is released, the selected range will be re-displayed to fill the current window. This operation can be repeated as often as required. Altering the Range of Both Axes Click and hold the left mouse button at one end of the region of interest and drag the cursor to the diagonally opposite corner. As the cursor is dragged, a "rubber band" is stretched out to indicate the region selected; do not go beyond the bounds of the axes. When the mouse button is released, the selected region will be re-displayed to fill the current window. This operation can be repeated as often as required. Setting Magnified Ranges Setting Magnification Ranges using the Mouse Setting Magnification Ranges using the Menu Bar Magnify Command To Magnify the Range of the Intensity Axis using the Tool Bar To Change the Magnification of a Particular Range Setting Magnification Ranges using the Mouse Click and hold the middle mouse button at one end of the region of interest and drag the cursor horizontally to the other end. As the cursor is dragged, a "rubber band" is stretched out to indicate the range selected; do not go beyond the bounds of the axis. When the mouse button is released, the selected range will be re-displayed with an initial magnification factor of two. Alternatively, pressing the Shift key while using the left mouse button will perform the same operation. Setting Magnification Ranges using the Menu Bar Magnify Command 1. Either: c. Select the Menu Bar Display, Range, Magnify command. Or: d. Double-click on the range magnification description of an existing magnified range. In either case, the Spectrum Magnify dialog is invoked. 2. Enter the magnification factor to be applied in the By text box. 3. Enter the range to be magnified in the From and To text boxes. 4. To define more than one magnification range on the displayed Spectrum, select a new range in the Range list box and repeat Steps 2 and 3. Up to five different magnified regions of the Spectrum can be defined. 5. Select the OK button to close the dialog. The Spectrum is re-displayed with the data in the selected regions magnified by the requested factor. The magnified regions are displayed in a different color and labeled with the magnification factor. Where multiple magnification regions have been defined, to select the current magnification range, click in the magnification description that appears above the range. The description will change color to red to indicate the currently selected range. To Magnify the Range of the Intensity Axis using the Tool Bar Select to increase the magnification of the current range. The current magnification factor is multiplied by 1.5, and rounded up to the nearest even number to give the increased magnification factor. If the initial magnification factor is 2, this will give subsequent magnification factors of 4, 6, 10, 16, etc. Select to decrease the magnification of the current range. The current magnification factor is divided by 1.5, and rounded down to the nearest even number to give the decreased magnification factor. If the initial magnification factor is 16, this will give subsequent magnification factors of 10, 6, 4, etc. To Change the Magnification of a Particular Range 1. Either: a. Select the Menu Bar Display, Range, Magnify command. Or: b. Double-click on the range magnification description of an existing magnified range. In either case, the Spectrum Magnify dialog is invoked, see Setting Magnification Ranges using the Menu Bar Magnify Command. 2. Enter the new magnification factor in the By text box. 3. Select the OK button. Deleting Magnification Ranges Select the Tool Bar button to delete the current magnification range. To delete all the magnification ranges: 1. Either: a. Select the Menu Bar Display, Range, Magnify command. Or: b. Double-click on the range magnification description of an existing magnified range. In either case, the Spectrum Magnify dialog is invoked, see Setting Magnification Ranges using the Menu Bar Magnify Command. 2. Select the Default button; this will delete all magnification ranges. 3. Select the OK button. Restoring the Display Selecting the Tool Bar button once restores the display to its previous state. Selecting it a second time restores the display to the default range. Note: These operations do not remove magnification ranges. Setting the Display Range Defaults Note: The display range default settings specify both the effects of selecting the Tool Bar button, and adding a new Spectrum to the display. More: To Change the Default Display Range To Change the Default Display Range 1. Select the Menu Bar Display, Range, Default command; the Default Spectrum Range dialog is invoked. 2. Make the required changes, see below. 3. Select the OK button. Default range Specifies whether the mass axis will range from the first peak to the last peak in the scan (Data), or over the range requested when the Frame acquisition started (Acquisition). Note: This frame is only relevant to Centroid mode acquisitions. Default graph If there is more than one spectrum in a window, this option specifies whether the mass range for that window is made large enough to Frame include the mass ranges of All the spectra, or large enough for the Current spectrum only. Automatic If this option is checked, the display range will return to the specified default (see Default range and Default graph above) when a new range default spectrum is added to a Spectrum Window. If this option is not checked, the display range will remain unchanged when a new spectrum is added. Displaying a Spectrum as a List The display in the current spectrum window can be replaced with a list of masses and intensities of the peaks in the currently selected spectrum. More: To Display a Spectrum as a List To Restore the Graphical Display To Print a Report of the Spectrum Listing To Display a Spectrum as a List Select the Menu Bar Display, List Spectrum command. A check mark is placed against the List Spectrum menu item. Most of the Menu Bar commands and the Tool Bar may still be used. To Restore the Graphical Display Select the Menu Bar Display, List Spectrum command. The check mark is removed from the List Spectrum menu item. To Print a Report of the Spectrum Listing 1. Select the Menu Bar File, Print Report command. The Spectrum Print Report dialog is invoked. 2. Select the Range of data to be displayed. Select Data to print a listing of the whole data file. Select Display to print a listing of the current display range. 3. Select the Header Information and Peak Information to be printed by selecting the relevant check boxes. 4. Select the OK button to exit and print the report. Controlling the Appearance of the Display General To Change the Display Parameters General Each Spectrum Window has its own set of Display Parameters, which determine the appearance of the Spectrum display. The parameters can be inspected and altered for the current Spectrum Window from the Spectrum Display dialog. To Change the Display Parameters 1. Select the Menu Bar Display, View command; the Spectrum Display dialog is invoked. 2. Make the required changes, see below. 3. Select the OK button. Normalize Data These controls specify the scale on the intensity axis. To Frame Largest Displays the largest peak currently on display at 100% of the intensity axis. Peak on Display Base Peak Displays the largest peak at 100% of the intensity axis. in Spectrum Mass When selected, 100% on the intensity axis represents the height of the peak at the mass specified in the adjacent text box. Intensity When selected, 100% on the intensity axis represents the intensity specified in the adjacent text box. Baseline at Scales the vertical axis from 0%. Zero Baseline When selected, the vertical axis is scaled from the intensity specified in the adjacent text box. This option can be useful for displaying spectra that have a raised baseline. Link Gives all axes in the current window a common vertical scale. This enables two spectra to be plotted on the same intensity scale, in Vertical order to overlay and compare them. Axes Data Threshold When processing centroid type data, it can be useful to specify an intensity threshold. Peaks whose intensity is less than the Frame threshold will not be displayed. This frame allows this to be done. Note: The threshold controls are not applicable to continuum mode data. % Full scale Sets the threshold as a percentage of the intensity of the largest peak in the spectrum. Intensity Sets an absolute intensity threshold. Style Frame Overlay Allows multiple traces in the same window to be superimposed on the same axis. Graphs If the option is not selected, the traces will be drawn on separate axes, arranged vertically. Note: When spectra are overlaid, only the currently selected trace is annotated. Graph Displays the graph header information at the top of the Spectrum. Header Process Displays process information in the Spectrum header. Description Note: The Graph Header option overrides the Process Description option, i.e. if the Graph Header is deselected, the Process Description will also be deselected. Component Displays a summary of the components identified so far on each spectrum. Table Show The Background Subtract process sets a zero level in continuum data, and in the resultant spectrum, half the noise lies below that Negative level. This option specifies whether these negative data points are Data displayed. If the option is selected, the scale on the intensity axis ranges from the smallest (most negative) intensity to the largest. If the option is not selected, the intensity axis ranges from zero to the largest intensity. Refer to The Background Subtract Process. Show Zero Draws a horizontal line to represent the zero level in the spectrum. Again, this is useful for gauging the effect of the Background Level Subtract process. Hide Lock For Mass Measured Tof data a Lock Mass Peak can be defined, this peak will be shown in a different color on the spectrum. This Mass Peaks option specifies whether the lock mass peak is displayed. If the option is selected, the Lock Mass Peak is not displayed. Refer to Lock Mass. Fill Trace Colors the area under the spectrum. Note: This option only applies to continuum-type (not centroid) data. This option is enabled when the Overlay Graphs control is Split Axis selected. It allows the User to alter the aspect ratio of the spectrum by dividing the mass axis into segments, then arranging the segments vertically. For example, if a spectrum of from 40 to 340 amu is on display, and 3 is selected in the Split Axis option, the display will show three axes: one from 40 to 140 amu, one from 140 to 240 amu, and one from 240 to 340 amu. This option is enabled when the Overlay Graphs control is Overlay selected. It allows the User to offset each subsequent spectrum Step X(%) trace by a percentage of the horizontal axis. This can make it easier to examine overlaid traces. This option is enabled when the Overlay Graphs control is Overlay selected. It allows the User to offset each subsequent spectrum Step Y(%) trace by a percentage of the intensity axis. This can make it easier to examine overlaid traces. Grid Enables the User to specify a grid to be displayed on the Spectrum display. The pattern of the lines that make up the grid can be chosen as Dot, Dash, or Solid. Select Off if no grid is to be displayed. Invokes the Header Editor, which allows editing of the header Header button information displayed at the top of the window. For more information, see The Header Editor Dialog . Controlling the Appearance of Peak Labels Each Spectrum Window has its own set of Peak Annotation Parameters, which determine the appearance of peak labels. The User can inspect and alter the parameters for the current window in the Spectrum Peak Annotation dialog. More: To Change the Peak Annotation Parameters To Annotate a Particular Peak To Change the Peak Annotation Parameters 1. Select the Menu Bar Display, Peak Annotation command; the Spectrum Peak Annotation dialog is invoked. 2. Make the required changes, see below. 3. Select the OK button. Annotation Type Frame Decimal Select between zero and four decimal places to be displayed on mass labels. Places Note: This control does not affect intensity labels, which are always displayed as integers. Mass Labels peaks in the current spectrum window with their masses to the specified number of decimal places. Intensity Labels peaks in the current spectrum window with their intensity as an integer value. Delta Mass Displays the difference between the mass of each peak in the spectrum and the specified mass. Note: The following controls are only applicable to ElectroSpray data. Mass Error Labels peaks in a MaxEnt Spectrum with their probable mass errors. Intensity Provides an error range on the intensity of MaxEnt Spectrum peaks. Error Component Labels peaks with the name of the appropriate component, and charge state if raw data is being viewed. (In Transform or MaxEnt Label spectra, peaks are labeled with component name alone.) Enables the Series button; this invokes the Ion Series Annotation Ion Series dialog, which allows the User to select which ion series to annotate Label (see the "Mass Spectral Fragments" section in the BioLynx & ProteinLynx User's Guide). Files with extensions ion, int and tab are created and stored in the raw data file when matching theoretical mass spectral fragment ions generated in BioLynx with centroided data. Digest Annotates the digest fragments matched from BioLynx, e.g. fragment T10-11. label Annotation Used to enter the minimum intensity for a peak to be labeled. Threshold frame % Full scale When selected, enter a value in the adjacent text box to define the threshold as a percentage of the base peak intensity. Intensity When selected, enter a value in the adjacent text box to define the threshold intensity. Level From the drop down list box, select the number of labels that appear on the chromatogram; this can be set to High, Medium or Low. To Annotate a Particular Peak Hold down the keyboard Ctrl key and right-click on the peak to be annotated. The peak will be mass labeled. To remove the mass label from the peak, hold down the Ctrl key and right-click on the peak a second time. Removing Spectra from the Display To Remove a Single Spectrum Trace from the Display To Remove Multiple Spectrum Traces from the Display Real-Time Display of Spectra Changing the Order of Displayed Spectra Adding Text to the Spectrum Display To Remove a Single Spectrum Trace from the Display 1. Press the keyboard Delete key. A dialog is invoked asking for confirmation of deletion of the currently selected spectrum trace. 2. Select the OK button; the dialog is closed and the selected traces are removed from the display. This operation does not affect the data stored on disk. To Remove Multiple Spectrum Traces from the Display 1. Select the Spectrum Menu Bar Display, Remove command. The Remove Spectra dialog is invoked. 2. The spectra in the current window are listed in the order in which they appear on the display. One or more spectra can be selected by clicking in the Spectra: list box. Clicking again on a selected item will cancel the selection. Selecting the All button selects all the spectra. 3. Select the OK button; the dialog is closed and the selected trace(s) are removed from the display. This operation does not affect the data stored on disk. Real-Time Display of Spectra If data are being acquired into a file, the associated spectra can be displayed in real time, by selecting the Tool Bar button; or the Spectrum Menu Bar Display, Real-Time Update command, which invokes the Spectrum Real-Time Update dialog. Each Spectrum Window has a separate real time update switch. The state of the switch for a particular Window can be ascertained by checking if the Tool Bar button is depressed, or by making that Window current, then selecting the Menu Bar Display menu. If real time update is enabled, the Real-Time Update item has a tick mark by it. Enable Enables the Real-Time update. Real-Time update Update Frame Latest scan When selected, displays the latest scan. Average all When selected, displays the average of all the scans acquired at present. scans Average When selected, displays the average of the latest number of scans defined in the text box. latest…scans Each spectrum window has a separate real time update switch. The state of the switch for a particular window can be ascertained by checking if the Tool Bar button is depressed, or by checking the state of the Spectrum Real-Time Update dialog Enable Real-Time update option. Changing the Order of Displayed Spectra When a window contains multiple traces, the order in which they are displayed can be changed. The spectrum that is first in the list is displayed at the bottom of the screen. Select the Spectrum Menu Bar Display, Move To First option to display the currently selected spectrum at the bottom of the screen. Select the Spectrum Menu Bar Display, Move To Last option to display the currently selected spectrum at the top of the screen. Adding Text to the Spectrum Display User text labels are added to a spectrum display in an identical manner to that for Chromatogram, refer to Adding Text to the Chromatogram Display for details. Exporting SEQUEST Files MassLynx has a facility to convert files into a format that can be used by the "SEQUEST" program. The "SEQUEST" program correlates uninterpreted tandem mass spectra of peptides with amino acid sequences from protein and nucleotide databases. It has been written by Jimmy Eng and John Yates (University of Washington). Note: This option is only enabled if BioLynx is installed. More: To Export a SEQUEST File To Export a SEQUEST File 1. Display the relevant centered MS/MS data file. 2. Select the Menu Bar File, Export SEQUEST file command. The Export SEQUEST compatible file dialog is invoked. 3. Make the required changes, see below. 4. Select the OK button. Precursor ion The precursor ion mass is picked up from the data file, if it mass: was entered in the Function Editor, otherwise, a value may be entered in this text box. This value defaults to 2, it may be changed as required. Precursor charge state: Partial Any known sequence information may be entered in this box. sequence information: File Path and The location and file name that the file will be saved to. The file name is the original file name, with the scan and function numbers Destination: appended to it. To change the destination, type a new destination in this box, or select the Browse button and select a new destination from the displayed dialog. Processing Spectra Several processes are available for use on spectra: , Refine, see The Refine Process. , Combine Spectra, see The Combine Spectra Process. , Background Subtract, see The Background Subtract Process. , Smooth, see The Smooth Process. , Center, see The Center Process. , Mass Measure, see The Mass Measure Process. , Integrate, see The Integration Process. More: Saving and Recalling Processed Spectra Saving and Recalling Processed Spectra The spectra resulting from any spectral processing can be saved with the raw data. More: To Save a Processed Spectrum To Reload Processed Data into Spectrum To Save a Processed Spectrum Select the processed spectrum in the Spectrum Window and select the Menu Bar File, Save Spectrum command. The Spectrum Save dialog is invoked, giving a brief description of the process being saved. Select the OK button to save the process and close the dialog. To Reload Processed Data into Spectrum 1. Select the Menu Bar File, Open command; the Spectrum Data Browser dialog is invoked. Note: The Spectrum Data Browser dialog is based on the standard MassLynx Data Browser dialog, see Opening Data Files: The MassLynx Window Data Browser Dialog for details. 2. Click on the raw data file from which the processed data was obtained. 3. Select the History button; the History Selector dialog is invoked, see The History Selector Dialog for details. 4. In the Process History: list, select the processed data to be loaded. 5. Select the OK button to exit the History Selector dialog. 6. Select the OK button to exit the Spectrum Data Browser dialog and load the processed data. The Refine Process The Refine process operates on centroid-mode data only. Its purpose is to automatically remove background ions from a spectrum thereby allowing it to be more easily identified, for example by library search. Select the Menu Bar Process, Refine command to invoke the Refine Spectrum dialog. A particular TIC peak is identified by specifying the peak top scan. The User supplies two parameters for the process; Window size and Noise threshold. The Refine algorithm proceeds by generating the summed mass chromatogram over a range of 1 Da centered on each integer mass in turn. It examines these chromatograms for a number of scans equal to the Window size around the peak top scan. (Window size is the half width in scans at baseline of the TIC peak of interest.) If there is a peak present in this range whose top-most point is within one scan of the peak top scan, and is more intense than the Noise threshold value, then this mass will appear in the refined spectrum. More: To Refine a Scan in a Centroid-Mode Data File To Refine a Scan in a Centroid-Mode Data File 1. Identify the scan at the top of the peak of interest. Display this scan in a spectrum window. This can be simply done by double-clicking on the peak in the Chromatogram Window. 2. Select the Spectrum Menu Bar Process, Refine command, the Refine Spectrum dialog is invoked. 3. Enter values for Window size and Noise threshold. For the first run, set Noise threshold to zero to show all peaks. 4. Select the OK button to start the process. 5. If the noise level in the refined spectrum is unacceptable, repeat the refine operation with a higher Noise threshold setting. Values in the range 0 to 10 are recommended. The current spectrum may also be refined, using the current refine parameters, by selecting the Spectrum Tool Bar button. The Combine Spectra Process The Combine Spectra process operates on centroid-mode or continuum data. Its purpose is to produce a single spectrum by subtracting averaged background spectra from the average of spectra from a TIC peak. The combined scan exhibits enhanced signal-to-noise and improved mass accuracy. The User specifies three scan ranges and a background factor. One range contains the scans across the peak top (the peak top scan range) and the other two ranges contain scans from the background, on each side of the peak. The scans across the peak top are averaged together and the average of all the background scans, multiplied by the background factor (X), is subtracted from the result. More: To Combine Scans in a Centroid-Mode Data File To Combine Scans in a Centroid-Mode Data File 1. Display the chromatogram peak of interest in a Chromatogram Window. 2. Select the Tool Bar button, or select the Menu Bar Process, Combine spectra command; the Combine Spectrum dialog is invoked. Note: The Combine Spectrum dialog may also be invoked from a Chromatogram Window by selecting the Chromatogram Menu Bar Process, Combine Spectra command. 3. Select the desired options (see below). 4. Select the OK button, the Combine Spectra process starts. Average Specifies the peak top scan range. This can be entered either by typing scan numbers separated by a colon (e.g. 619:626) in the text box, or by dragging across the peak using the right mouse button. Note: This field will only accept scan numbers in the range of the appropriate raw data file. Peak Specifies the maximum resolution of peaks in amu. This determines which peaks are to be regarded as being due to the separation same peak from scan to scan. Subtract Specifies the background scan range(s). These can be entered by typing scan numbers in the text box; each range must be in the form of two numbers separated by a colon. If there are two ranges, they must be separated by a comma (e.g. 606:612,631:637). Alternatively, the range can be entered by dragging across the peak using the right mouse button. Note: This field will only accept scan numbers in the range of the appropriate raw data file. Multiple Normally, when using the right mouse button to enter values, the first set of ranges is entered in the Average box and the second Average and third sets are entered in the Subtract box. Selecting the Multiple Average box changes this so that the first six sets of ranges are entered in the Average box, and the seventh and eighth are entered in the Subtract box. Specifies the background factor; a value of 1 is equivalent to X no scaling. Values less than or equal to zero will default to 1. Reset Clears the Average and Subtract text boxes. The Background Subtract Process Background Subtract adjusts the zero level in a continuum spectrum to lessen the effect of chemical noise caused by column bleed, etc. Both the Transform and MaxEnt processes rely on having background removed from the spectrum; MaxEnt, especially, will produce an inferior result if this is not done. On data with a curved background, typically ElectroSpray and FAB spectra, Background Subtract improves presentation and aids interpretation. A low order polynomial is fitted to the data to remove a constant, sloping or curved background from a spectrum. The algorithm fits a polynomial of specified order (zero is a flat baseline, one is a straight, sloping line, two is a quadratic shape, etc.) to a spectrum, such that a specified percentage (usually 30 to 50%) of the data points lies below the polynomial. This operation is performed to an arithmetical tolerance that is specified by the User. The Background Subtract process also gives the User the option to display a graph of the baseline, which will be fitted to the data before starting the process. More: To Subtract the Background from a Continuum Spectrum To Subtract the Background from a Continuum Spectrum 1. Select the Menu Bar Process, Subtract command; the Background Subtract dialog is invoked. 2. Select the desired options (see below). 3. Select the OK button, the Combine Spectra process starts. The Subtract status dialog box indicates the progress of the subtract algorithm. The convergence value in the dialog box is updated after every iteration. The algorithm terminates when convergence is less than tolerance. The User can choose whether to view the zero level and negative data in the spectrum by selecting the appropriate options in the Spectrum Display View dialog. Polynomial Specifies the order for the polynomial: 0 is a flat baseline, 1 is order a straight, sloping line, 2 is a quadratic shape, etc. Below curve Specifies the percentage of data points that lie below the (%) polynomial. The effect of increasing this parameter is to raise the zero level in the spectrum. The default value of 40% is based on the observation that around 80% of the data points in a typical ElectroSpray spectrum are noise, and only 20% signal. Half the noise lies above the zero line, and half below, therefore half of 80%, or 40% of the total number of data points, should lie below the background zero level. Tolerance The effect of increasing this parameter is to make the algorithm terminate sooner, but the result may not be as satisfactory. Flatten edges When selected, the software checks that the applied polynomial is flat or horizontal at the beginning and end of the trace. Make graph of Gives the User the option of seeing what the effect of the fitted Background Subtraction would be on the data before actually polynomial doing it. Select this option, then select the OK button. A graph of the polynomial function, which would be subtracted from the spectrum, is displayed above the resulting subtracted spectrum. If the Spectrum Display dialog Link Vertical Axes and Overlay Graphs options are selected, the new baseline will be superimposed on the existing data. When satisfied with the parameters being used, deselect the Make graph of fitted polynomial option. The Smooth Process Smoothing reduces the high-frequency noise present in a spectrum, thus aiding interpretation. It is strongly recommended that data is smoothed before mass measurement is attempted with the Center process, otherwise peaks may be created from the noise spikes. Note: Data for MaxEnt must not be smoothed. Three types of smoothing are implemented in MassLynx for smoothing spectra: , Moving Mean. , Savitzky Golay. , Moving Median. The most useful technique is Moving Mean. Using Savitzky Golay allows a heavier smooth without broadening the peak as much. Moving Median is used for removing noise spikes that are very much narrower than the real peaks (single ions, impulses from the electronics, etc.). All three smoothing methods slide a window along the data, averaging the data in the window to produce a point in the smoothed spectrum. The width of the smoothing window, in data points, is determined by the data system using the equation: , where m is the spacing between adjacent points on the mass axis, i.e., 0.0625 Da for raw continuum/MCA data, or equal to the value of the Resolution parameter for MaxEnt or Transform data. Moving Mean takes the arithmetical mean of the intensities of the data points in the window. Savitzky Golay takes an average of the intensities weighted by a quadratic curve. This tends to enhance quadratic-shaped features in the data (peaks). Moving median takes the arithmetical median of the intensities of the data points in the window. This process is unlike the previous two in that the median smooth iterates until the spectrum no longer changes. The effect is that the intensity of narrow spikes is reduced on successive iterations, to background level on convergence. More: To Smooth a Continuum Spectrum To Smooth a Continuum Spectrum 1. Expand a section of the spectrum sufficient to allow an estimate to be made of the width of a peak at half height. 2. Choose the Menu Bar Process, Smooth command, the Spectrum Smooth dialog is invoked, see below. 3. Set the Peak width (Da) parameter according to the value estimated in step 1, this can be done by dragging, using the mouse, over the peak at half height. 4. If Moving Mean or Savitzky Golay have been selected, the number of times the smooth is repeated may be changed, by changing the Number of smooths parameter from its default value of 2. Increasing this parameter gives a heavier smooth. 5. Select the OK button, the Smooth process starts. Peak width An estimate for this parameter may be obtained by dragging, (Da) using the mouse, over the peak at half height. Number of Specifies the number of times the smooth is repeated; increasing this parameter gives a heavier smooth. The maximum value is smooths 100. Note: The Number of smooths parameter has no effect on Median smoothing, which always iterates until the spectrum is unchanged. Smoothing method Frame Mean Selects the Moving Mean smoothing method. Median Selects the Median smoothing method. Note: The Median smoothing algorithm has the side effect of producing peaks with flattened tops. For this reason, it is recommended that a Median smooth be followed by a single iteration of a Mean or Savitzky Golay smooth. Savitzky Selects the Savitzky Golay smoothing method. Golay The Center Process Peak centering uses all the points across a peak in a continuum trace to calculate the mass of the peak center. The centering process can be used either to label each peak with the calculated mass, or to produce a single bar from each peak in a continuum spectrum. The calculation can be performed in three ways: , Select the most intense (top) point on the peak. This method is the least prone to errors caused by unresolved adducts in ElectroSpray spectra. , Calculate the centroid of the peak. This is equivalent to finding the vertical line passing through the center of gravity of the peak. This will provide a more accurate mass measurement, unless the peak contains unresolved adducts. , Calculate the median of peak area. This is equivalent to drawing the vertical line such that half the area of the peak lies on either side. There is little practical difference between the median and centroid methods, though it may be the case that the median is a more robust statistic on very asymmetric peak shapes. Masses from different experiments obtained by centering with different methods should not be compared. The centering algorithm looks for the trace rising then falling to indicate the top of a peak. The User specifies how many data points must be visible as a clear peak top before the algorithm turns the peak into a bar. For the centroid method, there is also the option of only using a specified fraction of the resolved part of the peak. This will help to avoid the mass given to the bar being affected by unresolved neighboring peaks. More: To Center a Continuum Spectrum To Center a Continuum Spectrum 1. Background Subtract the spectrum, see The Background Subtract Process. Background subtraction tells the centering algorithm how much of the spectrum is noise, and therefore reduces the amount of noise seen in the resultant bar spectrum. 2. Smooth the spectrum, see The Smooth Process. Smoothing will help the centering algorithm make sensible decisions about whether groups of data points represent peaks, or noise spikes. Note: MaxEnt spectra are an exception: they need centering to get an accurate mass just like any continuum spectrum. However, MaxEnt is designed to produce smooth spectra, and every peak in the MaxEnt result has already been interpreted by MaxEnt as significant. For this reason, neither smoothing nor subtraction of MaxEnt spectra is necessary prior to mass measurement. 3. Select the Menu Bar Process, Center command, the Spectrum Center dialog is invoked. 4. Select the desired options (see below). 5. Select the OK button, the Center process starts. Center method Frame Min peak An estimate for this parameter may be obtained by dragging, using the mouse, over the peak at half height; alternatively, enter a value width at half in the text box. height (channels) This parameter determines how many data points must be visible in the expected shape across the peak top, i.e. minimum width. For continuum/MCA data, setting this parameter to 4 is safe. Since there are sixteen data points collected per Dalton, the value 4 is equivalent to 0.25 Da. For MaxEnt results, the peaks can be very narrow. Sometimes there are two data points across the peak top. Therefore, for MaxEnt results, the only safe value for this parameter is 2. Too low a setting of this parameter will result in the centering algorithm producing bars from the high-frequency noise. Too high a setting of this parameter will result in the centering algorithm misinterpreting many peaks to produce a single bar. Top Selects the top method of processing. Centroid Selects the centroid method of processing. The fraction of the resolved portion of the peak that is used to calculate the centroid top may be changed, from its default value of 80%, in the adjacent text (%) box; recommended values are 60% to 95%. Median Selects the median method of processing. Centered spectrum Frame Create Creates a bar spectrum; the masses of the bars are calculated according to the selected center method. centered spectrum Heights When selected, the height of a bar represents the intensity of the continuum trace at the mass of the bar. Areas When selected, the height of a bar represents the sum of the intensities of the points across the peak in the continuum trace. Add Adds the bar spectrum to the current Spectrum Window. New Displays the bar spectrum in a new Window. window Replace The bar spectrum replaces the currently selected spectrum in the Window. Note: For Tof data this dialog will have an extra button. Select this button to display the QTOF Accurate Mass parameters dialog. For details, see QTOF Accurate Mass. The Mass Measure Process The Mass measure process performs a combination of background subtraction, smoothing and centering all in one command. Select the Spectrum Menu Bar Process, Mass Measure command to invoke the Mass measure dialog. Background subtraction takes place if the Background subtract control is checked. The Mass Measure dialog gives access to the Polynomial order and Below curve (%) parameters which are described in The Background Subtract Process. Mean Smoothing takes place if the Mean smooth control is checked. The Mass Measure dialog gives access to the Peak width, Number of smooths, Mean and Savitzky Golay parameters which are described in The Smooth Process. Peak Centering always takes place when the Mass measure process is used. The Mass Measure dialog gives access to the Min peak width at half height, Top and Centroid top parameters which are described in The Center Process. The Mass measure dialog always retains the last set of parameters used. Note: For Q-Tof data the Mass measure dialog will have an extra TOF button; this invokes the TOF Accurate Mass dialog. For details, see QTOF Accurate Mass. More: QTOF Accurate Mass QTOF Accurate Mass For Q-Tof data the Mass Measure dialog has a Use QTOF mass correction button which invokes the TOF Accurate Mass dialog. TOF Constants Frame Resolution Enter the resolution of the Mass Spectrometer. Np Enter a value for the Number of Pushes correction factor. multiplier Lock Mass Correction Frame Mass Determines the width of the mass window used to locate the lock mass data peak. The most intense peak in the range Lock Mass Window – Mass Window to Lock Mass + Mass Window is selected, and +/- mass correction based on this peak is performed. Lock Mass Specifies the reference lock mass, refer to Lock Mass. The TOF Transform Process Note: The TOF Transform process is only available if the BioLynx or ProteinLynx application manager has been installed. The TOF Transform process works on centroided (normally Q-Tof data). It both de-isotopes masses, and realigns to a single charge state mass axis. The TOF Transform dialog is invoked by the Spectrum Menu Bar Process, TOF Transform command. The values entered in the Min Molecular Mass and Max Molecular Mass text boxes specify the mass range over which the final output data will be aligned. For example, if the original spectrum has the largest mass of interest at 800 and the Max Charge State is 3, the mass range must be at least 2400 (3 x 800). The Max Charge State value should not exceed 4. In this example, if a mass at 700, with charge state 4, is present, it will not be seen (since 4 x 700 = 2800, and the specified mass range is 2400). The Integration Process The Spectrum integration process locates spectral peaks, draws baselines and calculates peak areas. Spectrum integration works over the full mass range of the spectrum. The assignment of baselines and separation of partially resolved peaks by verticals is determined by the Peak Detection parameters. For a detailed explanation of how the Peak Detection parameters affect integration see Integrating Chromatograms. To annotate the integrated spectrum with peak areas, select the Spectrum Peak Annotation dialog Intensity option, see To Change the Peak Annotation Parameters. More: To Integrate a Spectrum To Integrate a Spectrum 1. Select the Menu Bar Process, Integrate command. The Peak Detect dialog is invoked, refer to Standard Peak Detection Parameters for further information. 2. Edit the Peak Detection parameters as required. 3. Select the OK button to exit the dialog and perform the integration. The integration software will locate the peaks, draw baselines and calculate peak areas. ElectroSpray Data Processing In the ElectroSpray spectra of proteins, etc, each component produces a range of multiply charged ions in the original m/z spectrum. Therefore, additional processing must be performed to produce a molecular mass spectrum, also, due to the high accuracy required, a special calibration procedure is used. MassLynx provides two distinct methods for calculating the molecular mass spectrum: , Transform In this process, the User assigns charge states to peaks in the ElectroSpray m/z spectrum. This information is then used to transform the ElectroSpray data onto a molecular mass axis, see The Transform Process. , MaxEnt The MaxEnt algorithm uses the maximum entropy method to produce true molecular mass spectra from multiply-charged ElectroSpray spectra, see MaxEnt 1. More: Setting Adduct Mass for Transform and MaxEnt Finding Components for Transform Editing Components for Transform The Transform Process MaxEnt 1 MaxEnt Errors MaxEnt 2 MaxEnt 3 Setting Adduct Mass for Transform and MaxEnt Both the Transform and MaxEnt processes use the value for adduct mass in their calculations. To set the adduct mass value, select the Menu Bar Process, Set Adduct Mass command; this invokes the Set Adduct Mass dialog. The Adduct Type can be set to Hydrogen, Potassium or Sodium. Selection of more than one adduct type is not supported. Finding Components for Transform Initial Processing Finding Components To Find Components using the Manual Method; the Manual Find Components Dialog To Find Components when the Mass Range is Known; the Automatic Find Components Dialog Initial Processing Transform initially requires the assignment of charge states, and this is performed on a bar spectrum. Therefore, the first three steps are: 1. Background subtract the data, refer to The Background Subtract Process. In the Background Subtract dialog , suggested parameter values are: Polynomial order set to 1 for a flat baseline, or 5 for a curved baseline, Below curve set to 40%, and Tolerance set to 0.010. 2. Smooth the data with the Moving Mean algorithm, refer to The Smooth Process. The width of a peak in the raw data at half its maximum intensity must be measured; enter this value in the Spectrum Smooth dialog Peak width (Da) field. Set the Number of smooths parameter to 2. 3. Create a bar spectrum with the Center process, refer to The Center Process. Set the Spectrum Center dialog Min peak width at half height (channels) parameter to 4. Select Top as the centering method. Ensure the Create centered spectrum and Heights options are selected. It is convenient to put the bar spectrum into a new window, so it can be expanded to fill the Spectrum window when multiply-charged series are being identified; select the New window option to do this. Finding Components Multiply-charged series can now be identified as components. There are two methods of component identification: , The manual method is used when knowledge about the expected component mass is unknown. The User must identify two adjacent peaks in each series. MassLynx then identifies the rest of the series above the threshold and calculates the component's molecular mass, and the standard deviation associated with this mass. , The automatic method is used when knowledge about the expected component mass is known. It can be used to find each series in the spectrum in turn, or to identify all series in the spectrum. The disadvantage of this method is that a mass range to search over must be known in advance. Using a wide mass range may result in the false identification of spurious series. For the analysis of a true unknown, the manual method is preferred, so that the reliability of each entry can be checked. To Find Components using the Manual Method; the Manual Find Components Dialog The manual method for finding components uses the Manual Find Components dialog. Find Component Frame Peak 1 m/z These parameters are entered by typing their values directly in the and text boxes. Alternatively, after visually identifying a multiply Peak 2 m/z charged peak series, position the mouse pointer close to one peak in the series and right-click. Position the pointer close to an adjacent peak in the series and right-click again. The Peak 1 and Peak 2 controls will be updated to show the selected masses. Window… Specifies the tolerance on the position of each peak in the series. It may need to be increased from its default value of 0.5 Da for Da statistically poor data. Too low a value will result in the algorithm being unable to identify the whole of the series. Too high a value may result in the algorithm selecting wrong peaks. Dimers Allows correct charge assignment for the dimeric component in a monomer-dimer mixture. In this case, the monomeric series will obscure alternate peaks in the dimeric series. Therefore, to identify the dimer, the algorithm must assume a difference of two charge states rather than one between the two identified peaks. Threshold… Specifies a minimum intensity of peaks for the algorithm to consider. It is specified as a percentage of the intensity of the %BPI most intense peak in the spectrum. Reject… A molecular mass is calculated for each peak in the series. The mean molecular mass and standard deviation of that mean are then sd's calculated. This parameter offers the opportunity to discard any peak whose molecular mass is too far from the mean value. Such peaks are discarded and the mean is recalculated. This feature prevents outlying peaks from biasing the mean molecular mass measurement. The value is specified as a number of standard deviations in Da units. The default value of 2.00 means "Reject any peak whose molecular mass lies two or more standard deviations from the mean". Two is a safe value, as masses usually will be within two standard deviations of the mean. Set this parameter to a high value (e.g. 10.00) if this feature is not required. Delete Removes the current measured component. Calculates a component series and displays the result in the Found Measure component Frame. Found Displays the result of the component series calculation. component Frame Molecular The calculated component mass. Mass: Missing The expected masses of peaks that were not found by the calculation. peaks: OK Writes any processing carried out while the dialog is active is to disk. The component table will be modified and the currently active spectrum window will be updated to reflect any changes. Delete All Removes all the components for the particular scan. The components are not deleted from disk when the OK button is selected. To Find Components using the Manual Method: 1. Select the Menu Bar Process, Component, Find Manual command; the Manual Find Components dialog is invoked. 2. Having visually identified a multiply charged peak series, set the Peak 1 m/z and Peak 2 m/z, either directly, or by using the mouse, see above. 3. Select the Measure button. The Molecular Mass of the component will be displayed in the Found component frame. Also shown are the expected masses of peaks that were not found (Missing peaks:). 4. If the identification of the series is satisfactory, proceed to the next one. Otherwise, selecting the Delete button will remove the component from the component table, and the process may be repeated. 5. If required, select the Cancel button to abandon the process and exit the dialog box with no changes to the component table. To clear the component table completely, select the Delete All button. 6. Select the OK button, to complete the process. To Find Components when the Mass Range is Known; the Automatic Find Components Dialog The automatic method for finding components uses the Automatic Find Components dialog. Series Definition Frame Min Specifies the minimum number of peaks in the series. length… Peaks Max std Sets an upper limit on the spread of the molecular masses of the peaks in the series. The units are Daltons. dev… Da Peak Specifies the tolerance on the position of each peak in the series. It may need to be increased from its default value of 0.5 Da for window… statistically poor data. Too low a value will result in the algorithm Da being unable to identify the whole of the series. Too high a value may result in the algorithm selecting wrong peaks. The units are Daltons. Identify Specifies the number of largest peaks to be displayed, smaller peaks will not be displayed. largest… single peaks Allow Allows correct charge assignment for the dimeric component in a monomer-dimer mixture. In this case, the monomeric series will dimers obscure alternate peaks in the dimeric series. Therefore, to identify the dimer, the algorithm must assume a difference of two charge states rather than one between the two identified peaks. Peptide Specifies that rules for charge assignment will be made; this allows correct charge assignment for smaller molecules such as peptides. filter Mass Range Frame Min mol Specifies the lowest molecular mass that the algorithm can consider for a peak series. mass… Da Max mol Specifies the highest molecular mass that the algorithm can consider for a peak series. mass… Da Find Components Frame Threshold… Specifies the minimum intensity of peaks for the algorithm to consider. It is specified as a percentage of the intensity of the %BPI most intense peak in the spectrum. All Finds all components for the currently active spectrum, according to the specified parameters. First Finds only the first component for the currently active spectrum. The algorithm first considers peaks of highest intensity, then in descending intensity. After the first component is found and displayed, the First button changes to Next, allowing the next component to be found. OK Writes any processing carried out while the dialog is active is to disk. The component table will be modified and the currently active spectrum window will be updated to reflect any changes. Delete All Removes all the components for the particular scan. The components are not deleted from disk when the OK button is selected. To Find Components using the Automatic Method: 1. Select the Menu Bar Process, Component, Find Auto command. The Automatic Find Components dialog is invoked. 2. Set the Series Definition frame parameters, see above. 3. Set the Mass Range parameters. It is sensible to restrict the range as much as possible; the wider the mass range the algorithm is allowed to search over, the greater the chance of it making a series from peaks in the noise. 4. Set the Threshold parameter. A sensible threshold keeps the algorithm out of the noise, and helps to avoid the above problem. 5. Selecting the First button makes the algorithm find the best series containing the most intense unassigned peak. If no such series can be identified, then some of the parameters must be relaxed. First, check the Min length and Threshold parameters. If their values are reasonable, try larger values for Max std dev and/or Peak window. 6. Selecting the All button causes the algorithm to identify all component series present in the spectrum subject to the specified parameters. 7. If required, select the Cancel button to abandon the process and exit the dialog box with no changes to the component table. 8. To clear the component table completely, select the Delete All button. 9. When all components have been identified, select the Close button. Editing Components for Transform After the components present in the sample have been identified, the Edit Components dialog can be used to: , Rename a component. , Delete a component. , Sort and re-label the components in order of ascending molecular mass. , Add a component at known molecular mass, for instance singly-charged species. , Reject a single peak from the peak series. With poor data, this may improve the accuracy of the molecular mass. , Print a report showing all the peaks in the peak series for one or all components. More: The Edit Components dialog The Edit Component dialog To add a New Component at a Known Molecular Mass To Change the Name of a Component To Change which Peaks are used in the Calculation of a Component's Molecular Mass To Delete Components To Sort and Re-label the Components To Print the Peak Series for a Single Component To Print the Peak Series for All Components To Use a Component/Charge Delimiter The Edit Components dialog The Edit Components dialog is invoked by the Menu Bar Process, Component, Edit command. Add component Frame Molecular Specifies the molecular mass. mass Add Adds the component mass and label entered in the edit controls to the list box. Change label Frame Component Enter an identification label for the component (three characters maximum). label Update Uses the component label entered in the edit control to update the list box and the currently active spectrum window. Component / charge delimiter Frame None When selected, no delimiting character is placed between the component name and the corresponding charge in the spectral peak annotations. Delimiter Allows a delimiting character to be placed between the component name and the corresponding charge in the spectral peak annotations. For example, selecting the ':' character would annotate a peak in the style of Com:12. Any character is valid. Update Uses the delimiter label entered in the edit control to update the peak annotations in all spectrum windows. Edit When selected, the component currently highlighted in the list box is displayed in the Edit Component dialog which allows editing of the individual peaks, see The Edit Component dialog. Sort Sorts the components in the list box by mass and labels will be updated. The currently active spectrum window will be updated to reflect any changes. Delete Deletes the component currently highlighted in the list box from the list box. The currently active spectrum window is updated to reflect any changes, however, the component is only deleted from the component table if the OK button is selected. Delete All Deletes all components from the list box. The currently active spectrum window is updated to reflect any changes, however, the component is only deleted from the component table if the OK button is selected. Print Prints the currently selected component. Print All Prints all the components displayed in the list box. OK When selected, the component table is modified and written to disk. The currently active spectrum window will be updated to reflect any changes. The Edit Component dialog The Edit Component dialog is invoked by the Edit Components dialog, Edit button; it allows editing of the individual peaks. A particular component can be rejected, or included, in the series by selecting either the Reject or Include button respectively. Note: Only those peaks lying within the spectrum range when the component was added will be listed. To add a New Component at a Known Molecular Mass 1. Select the Menu Bar Process, Component, Edit command. The Edit Components dialog is invoked. 2. Enter the component's mass in the Molecular mass box. 3. Select the Add button. The component is inserted into the component table using the next available label. To Change the Name of a Component 1. Select the Menu Bar Process, Component, Edit command. The Edit Components dialog is invoked. 2. Select the component to be renamed in the list box. 3. Enter the new name for this component (three characters maximum) in the Component Label text box. 4. Select the Update button. To Change which Peaks are used in the Calculation of a Component's Molecular Mass 1. Select the Menu Bar Process, Component, Edit command. The Edit Components dialog is invoked. 2. In the list box, select the component whose peak series is to be to changed. 3. Select the Edit button. The Edit Component dialog is invoked; this displays the peak series for that component. The peaks that are included in the calculation of the molecular mass of that component are indicated by a check mark [x]. 4. To prevent a peak from being used in the calculation of the component's molecular mass, select the peak in the list box, then select the Reject button; the check mark is removed from the component. 5. To add a peak to the calculation of the component's molecular mass, select the peak in the list box, then select the Include button. 6. Select the OK button to close the dialog. To Delete Components 1. Select the Menu Bar Process, Component, Edit command. The Edit Components dialog is invoked. 2. Select the component to be deleted from the list box. 3. Select the Delete button; select the Delete All button to delete all the components. To Sort and Re-label the Components 1. Select the Menu Bar Process, Component, Edit command. The Edit Components dialog is invoked. 2. Select the Sort button. This will sort the components in order of ascending mass and re-label them, starting at A. To Print the Peak Series for a Single Component 1. Select the Menu Bar Process, Component, Edit command. The Edit Components dialog is invoked. 2. Select the component to be printed from the list box. 3. Select the Print button. To Print the Peak Series for All Components 1. Select the Menu Bar Process, Component, Edit command. The Edit Components dialog is invoked. 2. Select the Print All button. To Use a Component/Charge Delimiter A delimiter can be used to separate the component label from the charge on m/z spectra. 1. Select the Menu Bar Process, Component, Edit command. The Edit Components dialog is invoked. 2. Select the Delimiter: option and enter the required delimiter in the adjacent text box. 3. Select the Update button. The spectrum labels are updated to include the delimiter. The Transform Process When components have been identified in the spectrum, the data system can assign charge states to each peak. The Transform algorithm uses this information to display the m/z spectrum on a true molecular mass axis. More: To Transform an ElectroSpray Spectrum onto a Molecular Mass Axis; The Transform dialog To Transform an ElectroSpray Spectrum onto a Molecular Mass Axis; The Transform dialog The Transform dialog is invoked by the Menu Bar Process, Transform command. Min mol Specifies the lowest molecular mass that the algorithm can mass… consider for a peak series. Daltons Max mol Specifies the highest molecular mass that the algorithm can mass… consider for a peak series. Daltons Resolution… Specifies the resolution (in Da) between data points in the Da/channel Transformed spectrum. Cut at Frame Allows the User to specify how the m/z spectrum is to be divided up. Lowest When selected, regions of equal charge extend to midway point between identified peaks. Lowest point may produce a superior transform when not all the components in the spectrum have been identified, or the sample contains overlapping series. Mid point When selected, regions of equal charge are divided at the lowest point between identified peaks. To carry out a Transform: 1. Identify components in the spectrum as described in Finding Components for Transform. 2. Select the background subtracted continuum spectrum. 3. Select the Menu Bar Process, Transform command; the Transform dialog is invoked. 4. Set the parameters as required, see above. 5. Select the OK button, the Transform process will start. MaxEnt 1 The MaxEnt algorithm uses the maximum entropy method to produce true molecular mass spectra from multiply-charged ElectroSpray spectra. It has been successfully applied to biopolymers such as proteins and oligonucleotides. The algorithm has several distinct advantages over the Transform process: , MaxEnt automatically finds the molecular weights of the components in a protein mixture without any knowledge other than that they lie within a specified mass range. This can be large, e.g. 5 to 100 kDa. To reduce processing time, the technique currently involves a preliminary survey run, producing a coarse output to find the approximate masses of the components present. , The reconstructed MaxEnt spectrum exhibits enhanced resolution and signal-to-noise ratio. , The reliability of the result can be assessed by probabilistic methods. Thus, a probable error range can be calculated for each mass. , MaxEnt data are as quantitative as any ESMS data. The areas under the peaks in the MaxEnt profile spectrum are representative of the summed intensities of each component's multiply-charged series in the original m/z data. Transform works from the raw m/z data, combining the peaks from each component into a single peak on the molecular mass scale. Because several peaks in the m/z data are used to produce a single peak in the Transform, the Transformed spectrum exhibits better signal-to-noise than the raw data. However, the Transformed peaks are no better resolved than in the original data. MaxEnt retains the mass accuracy given by Transform on components that are adequately resolved in the original data. In addition, because of its ability to reveal resolution of peaks which is not apparent in the raw data, MaxEnt allows the mass measurement of components which were previously too poorly resolved for mass measurement in the transformed spectrum. MaxEnt finds the simplest molecular mass spectrum (spectrum of maximum entropy) that could account for the observed m/z data. The algorithm works iteratively; it takes an initial approximation to the molecular mass spectrum, and then uses programmed knowledge of chemistry and mass spectrometer physics (the damage model) to synthesize a corresponding m/z spectrum (the mock spectrum). It then compares the mock data to the observed (real) data, and uses the difference between the two to guide it to an improved molecular mass spectrum. The algorithm terminates when there is sufficiently little difference between mock and real data. A MaxEnt damage model describes the shape and width of the peaks in the observed m/z data, which is a composite of two effects. One effect is chemical; the distribution of molecular isotopes has a characteristic shape that is a function of molecular mass. The other effect is physical, caused by diffraction effects in the mass spectrometer. The latter effect, alone, can be seen by running a monoisotopic sample, for instance, Caesium Iodide. The current implementation of MaxEnt provides a single damage model. This is a Gaussian curve of constant width, which is a composite model of both of the above effects. To use this model, the width of a peak in the observed m/z data at half height must be measured. Note: The MaxEnt algorithm needs to accurately measure noise within a data file. For this reason, the Ion Counting Threshold should be set to zero when acquiring data that will be analyzed using MaxEnt, see the "MassLynx NT Guide to Data Acquisition" for further details. More: The MaxEnt 1 dialog To Produce a Survey Spectrum How to Establish the Correct Peak Width Parameter to Use When Processing Multiply_Charged Data by MaxEnt Interpreting the Survey Spectrum To Stop a MaxEnt Run Before the Algorithm Converges To Produce the Definitive MaxEnt Spectrum To Examine the Fit of Mock to Real Data Mass Measurement of MaxEnt Spectra To Produce a Profile Spectrum with Accurate Masses To Produce a Bar Spectrum with Heights Proportional to Component Concentration The MaxEnt 1 dialog The MaxEnt 1 dialog is invoked by the Menu Bar Process, MaxEnt 1 command. Output Mass Frame Ranges Specifies the output mass ranges. A single range is input as two numbers separated by a colon. Multiple ranges are separated by a comma. Specifies the resolution of the output. Resolution… Da/channel Damage Model Frame Uniform Selects the Uniform Gaussian damage model. Gaussian Width at Enter the appropriate value for this parameter in the text box. half For a detailed discussion on determining the correct value for height… this parameter, see How to Establish the Correct Peak Da Width Parameter to Use When Processing Multiply-Charged Data by MaxEnt. Simulated Selects the Simulated Isotope Pattern damage model. Isotope Pattern Enter the appropriate value for this parameter in the text box. Spectrometer Blur Width… Da Minimum The parameters in this frame place limits on the relative Intensity heights of adjacent peaks in the same series Ratios Frame Left… % Sets the limit for the relative heights of adjacent peaks at the low mass end of the spectrum. For example, if the parameter is set to 30%, and the most intense peak in the series is the 15+ peak, the 16+ peak must then be at least 30% as intense as the 15+ peak, the 17+ peak must be at least 30% as intense as the 16+ peak, etc. Right… % Sets the limit for the relative heights of adjacent peaks at the high mass end of the spectrum. For example, if the parameter is set to 40%, then the 14+ peak must be at least 40% as intense as the 15+ peak, the 13+ at least 40% as intense as the 14+, etc. Note: The default values of 33% for each of these parameters will always work, but, for most data sets, these values can profitably be increased. In particular, when doing a survey run, increasing the Left and Right Minimum Intensity Ratios will give significant reduction in the intensity of the "harmonic artifacts", i.e. the peaks at twice, three times, etc. the mass of each component. Completion options Frame Iterate to When selected, the MaxEnt process will continue iterating until it converges. convergence Maximum When selected, the MaxEnt process will perform up to the number of number of iterations specified in the adjacent text box. iterations Exit dialog When selected, MaxEnt will automatically accept the results, on exit the MaxEnt dialog, and display the MaxEnt spectrum on completion completion. If this option is not selected, the MaxEnt dialog will remain displayed on completion, giving the User the option to accept the results and save the MaxEnt spectrum, or discard the results. OK Starts the MaxEnt process. To Produce a Survey Spectrum The sole purpose of producing a survey spectrum is to determine the approximate masses of the components present. It is possible to analyze a complete unknown by selecting a very wide output mass range, e.g. 10 to 100 kDa. Usually, the major components are revealed after three or four iterations. 1. Background subtract the data, refer to The Background Subtract Process. In the Background Subtract dialog, set the parameters to fit an appropriate polynomial with 30 to 50% of the data below it. 30% usually leaves a low level of noise in the MaxEnt result; the User may wish to increase this for noisier spectra. 2. MaxEnt will process the data actually on display. Hence, the User can "rubber-band" the display to exclude parts of the spectrum that contain noise. This can improve the MaxEnt result in some cases. In addition, if the spectrum has a flat baseline, it is possible to remove this with the mouse, "rubber-banding" in the vertical direction. 3. Select the Menu Bar Process, MaxEnt 1 command, the MaxEnt 1 dialog is invoked, see The MaxEnt 1 dialog. 4. Set up the Output Mass range, in the Ranges text box. The mass range is given as two numbers separated by a colon, e.g. 10000:100000. 5. The Resolution parameter controls the "texture" of the result. Set this parameter to a value in the range 10 to 25 Da/channel. This will give a coarse result, not showing fine detail and without accurate masses, but the spectrum will suffice to locate the major components for a finer run over a smaller mass range. 6. Select the required damage model option in the Damage model frame. To use the Uniform Gaussian model, the average width at half height of a peak in the m/z spectrum must be estimated. For a detailed discussion on determining the correct value for the Width at half height parameter, see How to Establish the Correct Peak Width Parameter to Use When Processing Multiply-Charged Data by MaxEnt. 7. Set the Left and Right Minimum Intensity Ratio parameters. 8. Select the required options in the Completion options frame. 9. Select the OK button. The MaxEnt status dialog will appear. The algorithm will initialize itself, then draw molecular mass axes, and the first iteration will start. How to Establish the Correct Peak Width Parameter to Use When Processing Multiply-Charged Data by MaxEnt When processing data by MaxEnt, it is crucial that the correct peak width at half height is used. The only sure way to establish this width is to measure it, using peaks that are known to be singlets. The ideal way is to measure the width of a known singlet in the m/z spectrum to be processed. For example, in a haemoglobin spectrum, it may be required to separate and measure the components in an unresolved ,-globin doublet, when it is known that the ,-globin is a singlet. The measured width of an ,-globin peak near the center of the spectrum may then be used directly in the MaxEnt processing, since the molecular weights of the two globins are similar. In many situations, however, the peaks in the sample data will not be sufficiently resolved for their widths at half height to be measured. In these cases, it is necessary to measure the peak width from a multiply-charged spectrum run under identical conditions as the sample spectrum, and known to contain singlets. This can be the spectrum used to calibrate the sample spectrum, or another spectrum containing known singlets. In either case, it will generally be necessary to correct the measured peak width in order to find the value to use when processing the data by MaxEnt. This is derived as follows: Let the measured width at half height of a singlet in the 'calibration' spectrum be wc and let the peak have nc charges. Let the molecular weights of the 'calibration' compound and the sample be Mc and Ms respectively. Let the theoretical widths at half height due to the isotopic distribution of the elements in the molecule be Wc and Ws for Mc and Ms respectively. These may be found from the following figure. It is assumed that the width of a peak in the m/z spectrum is made up of two components; a component due to the theoretical isotopic distribution and a component due to the instrument itself (wi). These are assumed to be Gaussian, and are added as the root of the sum of the squares. Hence, wc2 = wi2 + (wc/nc)2 - - - - - - - - - - - - (1) and ws2 = wi2 + (Ws/ns)2 - - - - - - - - - - - - (2) where ws is the width required for processing the sample spectrum by MaxEnt, and ns is the number of charges on a peak at a similar part of the m/z spectrum to that used for measuring wc. Combining (1) and (2) to eliminate wi, ws2 = wc2 + (Ws/ns) 2 - (Wc/nc)2 - - - - - (3) Interpreting the Survey Spectrum The following figure shows the first three iterations of a MaxEnt survey run on a data set produced from leech haemoglobin. After the first iteration, the major components are already visible, but the harmonic artifacts at twice the mass of each component are present at significant intensity. Also, the background level is high, and rises with increasing mass. After the second iteration, the intensity of artifacts and background level has been greatly reduced. Neither is present with significant intensity after three iterations. Sub-harmonic artifacts at fractions (half, quarter, etc.) of the true molecular mass for the first couple of iterations may also be seen. First three iterations of a MaxEnt survey run on leech haemoglobin To Stop a MaxEnt Run Before the Algorithm Converges 1. Select the Halt button. 2. The result may now be accepted by selecting the OK button, or discarded by selecting Cancel. MaxEnt may also be restarted by selecting the Restart button. 3. If the spectrum is accepted and MaxEnt is to be started later, this may be done by selecting the Menu Bar Process, MaxEnt command again. To Produce the Definitive MaxEnt Spectrum Once the approximate masses of the major components are known, whether from prior knowledge of the sample, or a MaxEnt survey run, the definitive MaxEnt spectrum revealing all the fine structure can be produced: 1. Either select the background subtracted data used to produce the survey spectrum, or use Background Subtract to produce some. 2. Select the Menu Bar Process, MaxEnt 1 command; the MaxEnt 1 dialog is invoked. 3. Set up the Output Mass range from the knowledge of the approximate masses of the major components. The run time of MaxEnt is directly proportional to the number of data points in the output, and this number is the product of mass range and reciprocal of resolution. Therefore, do not set the mass range unnecessarily wide. 4. Note that two, or more, Output Mass Ranges separated by commas may be selected, e.g. 16500:17500, 24500:26500. Using this facility reduces the processing time. The Output Mass Ranges should include all the significant components found in the survey run, in order to make the definitive MaxEnt spectrum a faithful representation of the original data. 5. Set the Resolution parameter to 1.0 Da/channel. Generally, this is sufficiently small to ensure there will be several data points across each peak in the output, and a centroid can be taken to give an accurate mass. Occasionally, a smaller value e.g. 0.5 Da/channel may be necessary. This will increase the processing time, however. 6. Set the Damage model and Minimum intensity ratios parameters, as described above. 7. Select the OK button. To get a definitive result, MaxEnt must to run to completion. It will then produce two spectra; one is the MaxEnt result on a molecular mass axis, and the other is the mock data, explained in MaxEnt 1. Examining the mock data can help decide how good the parameter settings were. Mock data should fit the observed data within the tolerance of the noise. To Examine the Fit of Mock to Real Data 1. Select the Menu Bar Display, View command. The Spectrum Display dialog is invoked, see Controlling the Appearance of the Display. 2. Select the Style frame, Overlay Graphs option. This will cause spectra in the same window to be superimposed. 3. Select the Normalize Data to: frame, Link Vertical Axes option. 4. Select the OK button. 5. The raw data must now be displayed in the window containing the mock data. Click inside the window containing the mock data, then select the Menu Bar File, Open command. The Spectrum Data Browser dialog is invoked. 6. Ensure that the Add Data radio button is selected, then select the raw data from the list box. 7. Select the OK button. The Minimum Intensity Ratio parameters will affect the intensities of the peaks in the mock data, and the appropriate damage model width parameter will affect the widths of the peaks in the mock data. Mass Measurement of MaxEnt Spectra Special interpretation must be placed on peaks in MaxEnt spectra. The topmost point of the peak is not the most probable estimate of the peak's mass; rather, a centroid must be taken. The height of a MaxEnt peak is an indicator of how good an estimate the algorithm can make of the mass. This means the height is not proportional to the relative concentration of that component in the sample; but the area is. There are two ways to produce MaxEnt spectra with accurate masses. The first presents the profile spectrum labeled with accurate mass values. The second presents the spectrum as bars, with the height of each bar being proportional to the area of the peak in the profile data. To Produce a Profile Spectrum with Accurate Masses 1. Select the MaxEnt spectrum. 2. Select the Menu Bar Process, Center command. The Spectrum Center dialog is invoked, see To Smooth a Continuum Spectrum. 3. Set the Min peak width at half height (channels) parameter to 1. This will interpret the smallest, narrowest feature in the spectrum as a peak. If this does not produce the required result, the value of this parameter can be increased to group the narrower features together with the wider ones. 4. Select a Center method. Top is provided mainly for compatibility with the LAB-BASE data system. Centroid top (%) is the recommended method, since the parameter can be set to use the well-resolved part of the peak only, keeping clear of baseline effects. Recommended values for Centroid top(%) are in the range 70 to 90%. 5. Ensure the Centered spectrum frame, Create centered spectrum option is not selected. 6. Select the OK button. To Produce a Bar Spectrum with Heights Proportional to Component Concentration 1. Select the MaxEnt spectrum. 2. Select the Menu Bar Process, Center command. The Spectrum Center dialog is invoked. 3. Set the Min peak width at half height parameter as described above. 4. Select a center method as described above, e.g. Centroid top (%)=90. 5. Select the Centered spectrum frame, Create centered spectrum option. 6. Select the Centered spectrum frame, Areas option. 7. Select the OK button. MaxEnt Errors A probable error range can be calculated for the mass of each peak in the MaxEnt spectrum. This is done by sampling the distribution of possible spectra at about a dozen points near the most probable spectrum. Hence, the error analysis requires a further dozen iterations of the MaxEnt kernel, and for this reason, it is a separate process. More: To Calculate the MaxEnt Errors MaxEnt Initialization Errors To Calculate the MaxEnt Errors 1. Form a MaxEnt profile spectrum with accurate masses as described above. 2. Select the Menu Bar Process, MaxEnt errors command. The status dialog will appear, and the first cloud sample will commence. Twelve samples are performed in all, and after the last one, the spectrum is redisplayed with the errors. 3. Save the errors by selecting the Menu Bar File, Save command. Note: The MaxEnt errors will only be seen when the Mass Error parameter has been selected in the Spectrum Peak Annotation dialog, see Controlling the Appearance of Peak Labels. MaxEnt Initialization Errors Occasionally, an error message is displayed when MaxEnt is started: MaxEnt initialisation error –1, or MaxEnt initialisation error -2. These errors mean that there is not enough memory available to execute the current MaxEnt operation. In this case: 1. Close down MassLynx and any other Windows programs in order to free all available memory. 2. Run MassLynx again. 3. Load the spectrum to be processed. 4. Try to run MaxEnt again. 5. If the same error occurs, alter the MaxEnt parameters so that less memory is required. Reducing the mass range in the Ranges parameter, or increasing the Resolution parameter can do this. MaxEnt 2 MaxEnt 2 can be applied to any singly charged continuum spectrum to increase resolution and remove noise. For information about how the MaxEnt process works see MaxEnt 1. More: The MaxEnt 2 dialog To Use MaxEnt 2 To Stop a MaxEnt Run Before the Algorithm Converges The MaxEnt 2 dialog The MaxEnt 2 dialog is invoked by the Menu Bar Process, MaxEnt 2 command. Peak Width at Enter the appropriate value for this parameter in the text box. Half Height… Da Tolerance Increasing this parameter makes the algorithm terminate sooner, but the result may be inferior. The normal operating range for this parameter is between 0.030 and 0.300. Rate Controls the rate at which the MaxEnt Reconstruction process will run. It is recommended that values between 1.00 and 3.00 be used for this parameter. Overlay When selected, the new data calculated with each MaxEnt Iterations iteration is overlaid on top of the previous data. OK Starts the MaxEnt process. To Use MaxEnt 2 1. Display the singly charged continuum spectrum. 2. Adjust the display range to show the mass Range to be analyzed. MaxEnt will process the data range which is actually on display, this means that the display can be set to exclude parts of the spectrum which contain uninterpretable noise. 3. Select the Menu Bar Process, MaxEnt 2 command, the MaxEnt 2 dialog is invoked. 4. Set the parameters as required. 5. Select OK to start the analysis. The MaxEnt Reconstruction status dialog will appear. The algorithm will initialize itself, then draw molecular mass axes, and the first iteration will start. The status dialog shows the data produced by each iteration of MaxEnt. 6. When the MaxEnt reconstruction has finished the status dialog will display a message that the algorithm has converged. Select OK to accept the spectrum. MaxEnt will then produce two spectra; one is the MaxEnt result on a molecular mass axis, and the other is the mock data. The following figure shows part of a spectrum obtained from MALDI analysis of a peptide mixture. The upper trace shows background subtracted raw data, the middle trace shows background subtracted and smoothed raw data and the lower trace shows the MaxEnt reconstructed data. The MaxEnt reconstructed data shows much improved resolution of the isotope peaks. All MaxEnt processed spectra are stored to disk, with the raw data file; they can be selected using the Spectrum Data Browser, History button. Original data from Peptide mixture (upper and middle) and MaxEnt 2 data (lower) To Stop a MaxEnt Run Before the Algorithm Converges 1. Select the Halt button. 2. The result may now be accepted by selecting the OK button, or discarded by selecting Cancel. MaxEnt may also be restarted by selecting the Restart button. 3. If the spectrum is accepted and MaxEnt is to be restarted later, this may be done by selecting the Menu Bar Process, MaxEnt command again. MaxEnt 3 MaxEnt 3 can be applied to any low mass, multiply-charged continuum spectrum to resolve the multiply-charged peaks onto a singly-charged axis. The MaxEnt 3 program interprets isotope clusters to gain charge state information. For more information about how the MaxEnt process works see MaxEnt 1. More: The MaxEnt 3 dialog The MaxEnt 3 dialog (Tof Data) The MaxEnt 3 dialog (Quad Data) The MaxEnt 3 Advanced Parameters Dialog To Use MaxEnt 3 To Stop a MaxEnt 3 Run Before the Algorithm Converges The MaxEnt 3 dialog The MaxEnt 3 dialog is invoked by the Menu Bar Process, MaxEnt 3 command. Note: The MaxEnt 3 dialog format depends on the type of data selected. The MaxEnt 3 dialog (Tof Data) Min molecular Specifies the minimum mass for the output mass range. mass (Da) Max molecular Specifies the maximum mass for the output mass range. mass (Da) Maximum no of Defines the maximum charge state that MaxEnt can identify. charges For example, a value of 2 means that singly and doubly-charged peaks will be detected, 3 means singly, doubly and triply charged peaks will be detected, etc. Do not set this parameter to a higher value than needed, or artifacts may result. Peak width Frame Peak width Specifies the peak width at half height for the minimum mass 1 specified in the at m/z 1 text box. (Da)… at m/z 1 Peak width Specifies the peak width at half height for the maximum mass 2 specified in the at m/z 2 text box. (Da)… at m/z 2 Auto peak Determines peak widths automatically. width Note: determination The preceding Peak width options are disabled when this option is selected. OK Starts the MaxEnt 3 process. Advanced Invokes the MaxEnt 3 Advanced Parameters dialog, see The MaxEnt 3 Advanced Parameters Dialog. The MaxEnt 3 dialog (Quad Data) Min molecular Specifies the minimum mass for the output mass range. mass (Da) Max molecular Specifies the maximum mass for the output mass range. mass (Da) Maximum no of Defines the maximum charge state that MaxEnt can identify. charges For example, a value of 2 means that singly and doubly-charged peaks will be detected, 3 means singly, doubly and triply charged peaks will be detected, etc. Do not set this parameter to a higher value than needed, or artifacts may result. Peak width Specifies the peak width at half height. (Da) Advanced Invokes the MaxEnt 3 Advanced Parameters dialog, see The MaxEnt 3 Advanced Parameters Dialog. The MaxEnt 3 Advanced Parameters Dialog The MaxEnt 3 Advanced Parameters dialog is invoked by the MaxEnt 3 dialog Advanced button. No. of Specifies the number of ensemble members. The Ensemble recommended setting is 1. Members Iterations per Specifies the number of Iterations per Ensemble Member. Ensemble The recommended setting is 10, or 20. Member Compress Data Compresses the spectrum to half its original size, thus reducing run time. No deterioration in results has been observed as a result of using this option. MaxEnt 3 uses an ensemble of processes, notionally working in parallel. Increasing the No of Ensemble Members may improve results, but will increase run time. The Iterations per ensemble member parameter is a guide to the amount of CPU time each ensemble member is allowed. Again, increasing this parameter may result in improved results at the expense of runtime. To Use MaxEnt 3 1. Display the multiply charged continuum spectrum. MaxEnt will process the data range on display, and the display can be "rubber-banded" vertically to set a noise level. 2. Adjust the display range to show the mass Range to be analyzed. MaxEnt will process the data range which is actually on display; this means that the display can be set to exclude parts of the spectrum which contain uninterpretable noise. 3. Select the Menu Bar Process, MaxEnt 3 command; the MaxEnt 3 dialog is invoked. 4. Set the parameters as required. 5. Select the Advanced button to set the Ensemble parameters and the data compression option in the MaxEnt 3 Advanced Parameters dialog. 6. Select OK to start the analysis. The MaxEnt 3 status dialog will appear, showing the progress of the process. Select the Cancel button to stop a MaxEnt 3 run before the end of processing. 7. When the MaxEnt 3 has finished, MaxEnt will produce two spectra; one is the MaxEnt result on a molecular mass axis, and the other is the mock data. The following figure shows part of a spectrum obtained from TOF analysis of Glu-fibrinopeptide. The lower trace shows raw data, and the upper trace shows the MaxEnt 3 data. The MaxEnt data shows the charge state of the isotope peaks interpreted correctly. Original data from Glu-fibrinopeptide (lower) and MaxEnt 3 data (upper) All MaxEnt processed spectra are stored to disk, with the raw data file, and can be selected using the Spectrum Data Browser, History button. To Stop a MaxEnt 3 Run Before the Algorithm Converges 1. Select the Cancel button 2. The result may now be accepted by selecting the OK button, or discarded by selecting Cancel. MaxEnt may also be restarted by selecting the Restart button. 3. If the spectrum is accepted and MaxEnt is to be started later, this may be done by selecting the Menu Bar Process, MaxEnt command again. Isotope Cluster Abundance Plots MassLynx can produce an isotope cluster abundance plot for a given formula. For example, the following figure shows the predicted isotope model for the formula C9H10N2O2Cl2 (Linuron). Typical Isotope modeling display More: The Isotope modelling Dialog The User-definable elements Dialog To Produce an Isotope Cluster Abundance Plot The Isotope modelling Dialog The Isotope modelling dialog is invoked by the Menu Bar Tools, Isotope Model command, or the Elemental Composition Window Menu Bar Process, Set Isotope Match Parameters command, see The Elemental Composition Window Menu Bar. Formula Enter the chemical formula for the compound, using standard IUPAC (International Union of Pure and Applied Chemistry) notation. A formula can also be transferred from the BioLynx Protein or Nucleic Acid Editors by selecting the Elemental Composition Window Tool Bar button to transfer the molecular formula to the Windows Clipboard. The formula can then be pasted into the Isotope modelling dialog Formula text box by selecting the Paste button. Spectrum Modelling Frame Create Creates a continuum spectrum as well as the centroided Continuum spectrum. spectrum Peak width Enter a value for the continuum spectrum. at half-height Create When selected, the modeled spectrum will contain the multiply charged series that are present in ElectroSpray ElectroSpray spectra. spectrum Isotope cluster parameters Frame Separation Determines the resolution of the modeled spectrum. Peaks that would be closer together than this value are combined into a single peak. Min Determines the threshold below which peaks are not Abundance considered significant. (%) Molecular This frame contains parameters that determine the range Mass Range used for the peaks to be displayed in the modeled spectrum. Frame Full Range Selects the full range. Mass From Enter a value for the lower end of the range. Mass To Enter a value for the upper end of the range. Multiply-charge d ion Frame Multiply- Creates a single multiply-charged peak with the number of charged ion charges specified in the Charge state text box. OK Updates the isotope structure and initializes the isotope modeling algorithm. Paste Pastes a formula from the Windows clipboard into the Formula text box. Add Adds the spectrum to the current Spectrum Window. Replace The spectrum replaces the currently selected spectrum in the Spectrum Window. New window Displays the spectrum in a new Window. User elements Invokes the User-definable elements dialog, see The User-definable elements Dialog. Print masses Prints out the current range of masses. The User-definable elements Dialog The User-definable elements dialog is invoked by the Isotope modelling dialog, User elements button. It allows the User to specify a list of elements, isotopes, and/or molecules. Name Enter a name for the element in this text box. Symbol Enter a symbol for the element in this text box. Mass Enter a mass for the element in this text box. OK Closes the dialog and returns to the Isotope modelling dialog. Add Adds the current group to the list. A maximum of ten groups may be added. Deletes Deletes the currently selected group from the list. Update Updates the currently selected group with changed details from the Name, Symbol, or Mass text boxes. To Produce an Isotope Cluster Abundance Plot 1. Select the Spectrum Menu Bar Tools, Isotope Model command, or the Elemental Composition Window Menu Bar Process, Set Isotope Match Parameters command; the Isotope modelling dialog is invoked. 2. Set the parameters as required, see above. 3. Select the OK button. Elemental Composition Elemental Composition processes either a single or a multiple number of molecular masses, such that the elemental composition of the molecule(s) may be estimated. More: The EleComp Parameters Dialog To Produce an Elemental Composition Report To Update an Elemental Composition Report The Elemental Composition Window The Elemental Composition Window Menu Bar The Elemental Composition Window Tool Bar The EleComp Parameters Dialog The EleComp Parameters dialog is invoked by the Menu Bar Tools, Elemental Composition command. Use Single Select a single mass by entering the value in the text box, or Mass by right-clicking on a peak in the display. Use Multiple Selects the set of masses currently displayed. Masses OK Invokes the Elemental Composition dialog and an elemental composition report is generated from the selected peak(s). To Produce an Elemental Composition Report 1. Select the Menu Bar Tools, Elemental Composition command; the EleComp Parameters dialog is invoked. 2. Set the parameters as required, see above. 3. Select the OK button; an Elemental Composition Report is created and displayed in the Elemental Composition window, see The Elemental Composition Window. When the Elemental Composition Window is displayed, there are two other ways of generating reports: , A Spectrum list can be copied from another Windows application and pasted into the Elemental Composition window by selecting the Tool Bar button, or selecting the Menu Bar Edit, Paste command. The software will automatically generate a new report. , Selecting the Elemental Composition window Tool Bar button, or selecting the Menu Bar Process, Enter Single Mass command will invoke the Mass dialog. Enter a new Mass, or select a previously entered mass from the drop down list box. Select the Display all option to display all the results found for a mass. To display a limited number of results per mass, deselect the Display all option and select the arrows to change the value in the number box. For example, if 5 is entered, the five closest results to the mass will be displayed. If the Match isotopes option is selected, the Edit Parameters button is enabled; this invokes the Isotope Cluster Parameters dialog. Enter values for the Separation and Min. Abundance (%). Note: The Isotope Cluster Parameters dialog may also be invoked by the Elemental Composition window Menu Bar Process, Set Isotope Cluster Parameters command. Select the Mass dialog OK button to generate the Elemental Composition Report. To Update an Elemental Composition Report The search details for a report can be changed by selecting the Elemental Composition window Tool Bar button, or by selecting the Menu Bar Process, Set Parameters option. This invokes the Parameters dialog; change the required details and select the OK button. For more information on the Parameters dialog, see Elemental Composition Parameters. The Elemental Composition Window The Elemental Composition window is invoked, via the EleComp Parameters dialog, from the Spectrum Menu Bar Tools, Elemental Composition command, see To Produce an Elemental Composition Report. The window is split into three panes: , The top pane shows details of the number of masses used (if there are more than one) and the number of possible compounds found. , The bottom pane displays the spectrum of the components found. , The middle pane shows the following details for the compounds found: Mass The mass being analyzed. Note that for multiple masses, each mass will have a set of results. RA The percentage relative abundance; this is determined by expressing the intensity of the mass as a percentage of the intensity of the most intense peak. This is only displayed for multiple mass calculations. The calculated mass for the formula shown in the Formula column, Calc Mass subject to the specified tolerance limits. mDa The difference between the calculated mass and the entered mass, in milliDaltons. PPM The difference between the calculated mass and the entered mass, in parts per million. The double bond equivalent for the formula shown in the Formula DBE column. Formula A suggested formula for the entered mass. The formula is not a true atomic formula; it is merely a summation of the quantities of elements, isotopes, and/or superatoms that may compose the sample. In the formula, each symbol is displayed followed by the number of times of its occurrence. The Score relates to the fit of the reconstructed isotopic spectrum Score for each hit, to the original spectrum. The score is given as a rank, with a rank of 1 corresponding to the best match to the original spectrum. If the rank is given as "n/a", the reconstructed isotopic spectrum contains no masses in the mass range of the original spectrum. A symbol column is created in the middle pane for each Symbol columns element/isotope/superatom included in determination of possible elemental compositions. The value that appears in the symbol column on any given row is the number of occurrences of that element in the corresponding elemental composition as shown in the Formula column. Clicking on the Mass column heading will list the masses in reverse order. Clicking on any of the other column headings will display the values in ascending order for each mass, clicking a second time will display them in descending order for each mass. Holding the mouse cursor over the RA, DBE and symbol column headings will display the minimum and maximum values defined on the Parameters dialog. Holding the mouse pointer over the mDa and PPM column headings will display the tolerance values defined on the Parameters dialog. A status bar is located at the foot of the main application window. When masses are not currently being processed, the text aligned to the left of the bar simply says "For Help, Press F1". During processing, it displays processing information; for many of the larger spectra, the calculation may take several minutes to complete. A sunken pane, to the right of the progress text, displays the last mass that is to be processed. It is impossible to determine in advance how long the process will take, due to the nature of the formulae calculations. Therefore, a progress bar cannot be provided. However, by displaying the final mass to be processed, the User can determine approximately how long the calculations will take and therefore make the decision as to whether to terminate the process, if required. The Elemental Composition Window Menu Bar The Elemental Composition Window File Menu The Elemental Composition Window Edit Menu The Elemental Composition Window View Menu The Elemental Composition Window Process Menu The Elemental Composition Window Help Menu The Elemental Composition Window File Menu Saves the results in a plain text file (*.txt). A standard Save As Save Results dialog is invoked. Load Settings Loads a previously saved Parameter file. Select the required *.els file from the invoked Load Settings dialog. The software will automatically generate a report using these settings. Saves a Parameter file. Enter a name in the invoked Save Save Settings Settings dialog and select the Save button. Print Results Sends the table of results to the printer. Print Preview Standard Windows command. Print Setup Standard Windows command. Exit Closes the Elemental Composition Window. The Elemental Composition Window Edit Menu Copy If the top or middle pane is active the current set of results is copied to the clipboard. The format is copied as plain text and formatted in columns in the exactly the same manner as it is saved (see above). If the bottom pane is active, the spectrum on display is copied to the clipboard. Paste Pastes a mass spectrum that has been copied to the clipboard, into the application. The application then processes the data and generates the results, which are displayed in the results table. This button is disabled if there is nothing on the clipboard. If the clipboard data isn't useable spectrum data, no processing is carried out. Clear Results Deletes the current set of results in the table and resets the information window. The Elemental Composition Window View Menu Toolbar Toggles the Tool Bar on and off. Status Bar Toggles the Status Bar on and off. Toggles the Formula column on and off. Formula Table Toggles the symbol columns on and off. The Elemental Composition Window Process Menu Invokes the Mass dialog; see To Produce an Elemental Enter Single Composition Report. Mass Invokes the Parameters dialog; see Elemental Composition Set Parameters Parameters" section. Invokes the Isotope Cluster Parameters dialog; see To Produce Set Isotope an Elemental Composition Report. Cluster Parameters The Elemental Composition Window Help Menu Help Topics Invokes the MassLynx Help function. About Displays information about Elemental Composition. Elemental Composition The Elemental Composition Window Tool Bar Purpose Tool Menu equivalent Bar button File, Save Results Saves the Elemental Composition Report. Edit, Copy Copies the selection onto the clipboard. Edit, Paste Pastes the contents of the clipboard into the Elemental Composition Window. Clears the results from the Elemental Edit, Clear Results Composition Window. File, Print Prints the Elemental Composition Report. Invokes the Mass dialog. Process, Enter Single Mass Invokes the Parameters dialog. Process, Set Parameters Invokes the Isotope modelling dialog. Process, Set Isotope Match Parameters Elemental Composition Parameters The Parameters dialog is invoked by selecting the Elemental Composition Window Tool Bar button, or selecting the Menu Bar Process, Set Parameters option. The dialog has two pages: , General Parameters. , Symbol Parameters. Selecting the Reset to Defaults button will set the Hot Symbols (see Specifying Hot Symbols) and all other fields on the Parameters dialog to the default values. More: The Parameters Dialog; General Parameters Page The Parameters Dialog; Symbol Parameters Page Superatom Tables Superatom Limits The Parameters Dialog; General Parameters Page Tolerance Frame Expresses the parameters that comprise the tolerances for subsequent calculations as milliDaltons (mDa). MilliDaltons: PPM: Expresses the parameters that comprise the tolerances for subsequent calculations as parts per million (PPM). Note: 1. The above two options express the same thing. However, it is sometimes more convenient to use one rather than the other. Both are used to indicate how large a tolerance to allow when the cumulative mass of any given composition formula has been calculated. 2. The calculations use plus and minus this value, so the default 200 mDa and 5 PPM represent 200 mDa and 5 PPM. Minimum % Specifies the minimum relative abundance, i.e. specifies that only masses with a relative abundance above the specified value are to RA: be processed. The Results controls specify the quantity of results to display. Results Frame They are useful for reducing the time it takes to tabulate the results immediately following calculation. Display only Causes only those masses that fit within the specified tolerance parameters to be displayed. Otherwise, invalid masses are also valid results listed with "-" in the various columns to indicate that they have no results. No. Results When selected, up to 1000 results will be displayed for each mass, if they comply with the specified tolerance parameters. If this to option is not selected, the number of results displayed will be no display: greater than the value specified in the adjacent text box. The Double Bond Equivalent (DBE) specifies the Maximum: and Double Bond Minimum: number of double bonds per molecule considered to be Equivalent acceptable in producing a valid formula. A simple calculation can Frame be performed which will indicate how many double bonds there would be within a molecule of any given formula. The result of this calculation is then used to determine whether a given result falls within the specified validation limits. The mass modes utilize the Monoisotopic, Chemical, or Nominal Mass Mode mass of the active symbols when performing the calculations. Frame The Electron State specifies the preferred types of molecule to be Electron State included or excluded from the results. Frame Both odd Includes both odd and even electron types. and even Odd Includes odd electron types only. electron only Even Includes even electron types only. electron only The Parameters Dialog; Symbol Parameters Page Element Limits Up to ten "Hot Symbols" may be specified in this frame, to Frame allow their properties to be changed quickly and easily. Any element, isotope, or superatom can be specified as a Hot Symbol as shown in Specifying Hot Symbols. Check box Selects a Hot Symbol. Symbol Invokes the Select Hot Symbol dialog, see Specifying Hot button Symbols. From The minimum number of elements or isotopes that the calculated formula must contain; e.g. if the From value for Cl is 2, the formula must contain Cl2, but can contain any number above this, e.g. Cl3, Cl4, etc. The To value is the maximum number of elements or isotopes that To the calculated formula must contain; e.g. if the To value for Cl is 2, the formula must contain Cl2, but can contain any number below this. Note: If the From and To values are the same, the calculated formula must contain this exact number of elements or isotopes; e.g. if the From and To values for Cl are both 2, then the formula must contain Cl2. Periodic Invokes the Periodic Table dialog, see To Select an Table Element. Deselect Deselects all the Hot Symbols. All Superatoms Frame Tables Invokes the Superatom Tables dialog, see Superatom Tables. Limits Invokes the Superatom Limits dialog, see Superatom Limits. Note: This button is disabled if no Superatom Table has been loaded, see Superatom Tables. More: Specifying Hot Symbols To Select a Hot Symbol To Select an Element To Select an Isotope To Deselect an Element To Deselect an Isotope To Change the Minimum and Maximum Values Specifying Hot Symbols Hot Symbols may be specified in the Parameters dialog, Symbol Parameters page Element Limits frame, to allow their properties to be changed quickly and easily. Any element, isotope, or superatom can be specified as a Hot Symbol as follows: 1. Select the box next to the required symbol. 2. Select the Symbol button; the Select Hot Symbol dialog is invoked. 3. Select the box for the required element, or isotope. The current element, or isotope, is displayed in the Current Symbol frame. Note: By default, the list is sorted according to atomic number of the symbol. This can be changed to alphabetical order by clicking on the Symbol column header. Clicking on the Atomic No. column sorts back by atomic number again. Note that in alphabetical order, isotopes are still sorted numerically. 4. Select the OK button; the Select Hot Symbol dialog is closed. The name on the Parameters dialog, Symbol Parameters page, Symbol button will change to that selected in the Select Hot Symbol dialog. To Select a Hot Symbol 1. Check the box next to the required symbol. 2. If required, enter new values in the From and To boxes for the relevant symbol. To Select an Element Some common elements appear by default in the Parameters dialog, Symbol Parameters page; to select one of these, select the relevant box. To select an element that does not appear on the dialog: 1. Select the Periodic Table button; the Periodic Table dialog is invoked. Note: Selected elements are displayed in blue on the Periodic Table. 2. Click on the required element to invoke the Elements & Isotopes dialog; this contains a list of isotopes for the element. 3. Check the first box in the list and select OK on each dialog until the display returns to the Elemental Composition Window. Note: An element and its isotopes cannot be selected simultaneously; selecting the element will deselect the isotopes. To Select an Isotope 1. Select the Periodic Table button; the Periodic Table is invoked. Note: Elements with selected isotopes are displayed in blue on the Periodic Table. 2. Select the required element; the Elements & Isotopes dialog is invoked. This contains a list of isotopes for the element. 3. Select the boxes for the isotopes required, or select the Select Isotopes button to select all the isotopes. 4. Select the OK button on each dialog until the display returns to the Elemental Composition Window. Note: An element and its isotopes cannot be selected simultaneously, selecting the element will deselect the isotopes. To Deselect an Element Some common elements appear in the Parameters dialog, Symbol Parameters page; to deselect one of these deselect the relevant box, or select the Deselect All button to deselect all the elements. To deselect an element that does not appear in the dialog: 1. Select the Periodic Table button; the Periodic Table dialog is invoked. 2. Select the required element; the Elements & Isotopes dialog is invoked. 3. Deselect the first box in the list. 4. Select the OK button on each dialog until the display returns to the Elemental Composition Window. To Deselect an Isotope 1. Select the Parameters dialog, Symbol Parameters page, Periodic Table button; the Periodic Table dialog is invoked. 2. Select the required element; the Elements & Isotopes dialog is invoked. 3. Deselect the boxes for the isotopes that are not required. 4. Select the OK button on each dialog until the display returns to the Elemental Composition Window. To Change the Minimum and Maximum Values For the elements displayed in the Parameters dialog, Symbol Parameters page, enter new values in the From and To boxes for the relevant element. For other elements and for isotopes not displayed in the Parameters dialog: 1. Select the Periodic Table button; the Periodic Table dialog is invoked. 2. Select the required element; the Elements & Isotopes dialog is invoked. 3. Click anywhere on the row, then on the Minimum or Maximum value, as required, and enter a new value. The Minimum value is the minimum number of elements or isotopes that the calculated formula must contain; e.g. if the From value for Cl is 2 then the formula must contain Cl2, but can contain any number above this, e.g. Cl3, Cl4, etc. The Maximum value is the maximum number of elements or isotopes that the calculated formula must contain; e.g. if the To value for Cl is 2 then the formula must contain Cl2, but can contain any number below this. If the Minimum and Maximum values are the same then the calculated formula must contain this exact number of elements or isotopes; e.g. if the Minimum and Maximum values for Cl are both 2, then the formula must contain Cl2. 4. Select the OK button on each dialog until the display returns to the Elemental Composition Window. Superatom Tables A Superatom Table is an Access database containing details of large molecules that can be used in the elemental composition search in the same way as elements and isotopes. The elements database is loaded automatically on MassLynx start-up. The aminoacids.mdb database is also supplied with MassLynx; it contains details of twenty common amino acids. More: To Load a Superatom Database To Create a New Superatom Database To Load a Superatom Database 1. Select the Parameters dialog, Symbol Parameters page, Superatoms frame, Tables button; the Superatom Tables dialog is invoked. When this is first invoked, no database is loaded and the list box will be empty. 2. Select the Add Superatom Table button; the Load Superatom Database browser is invoked. 3. Select the required database file. 4. Select the OK button; the Load Superatom Database browser is closed and the database file is added to the Superatom Tables dialog. To Create a New Superatom Database Microsoft Access can be used to create new Superatom database tables, using the aminoacids.mdb file as a template. Note: The table name, column headings and data types must match those in aminoacids.mdb. Superatom Limits Select the Parameters dialog, Symbol Parameters page, Superatoms frame, Limits button to invoke the Superatom Limits dialog. All superatoms are automatically switched on by default when a database is loaded. They are all listed together in the order that the databases were loaded. If there is no more room left to accommodate some or all of the symbols, they are not switched on. A warning is displayed telling the User exactly which symbols could be enabled and how many currently enabled symbols will have to be switched off in order to turn on the superatoms. A similar message will also be displayed if an attempt is made to switch on elements or isotopes when the maximum number of displayable symbols has been reached. More: To Select a Superatom To Deselect a Superatom To Change the Minimum and Maximum Values To Select a Superatom 1. In the Superatom Limits dialog, select the boxes for the superatoms required, or select the Select All button to select all superatoms. 2. Select the OK button on each dialog until the display returns to the Elemental Composition Window. To Deselect a Superatom 1. In the Superatom Limits dialog, deselect the boxes for the superatoms not required, or select the Deselect All button to deselect all superatoms. 2. Select the OK button on each dialog until the display returns to the Elemental Composition Window. To Change the Minimum and Maximum Values 1. In the Superatom Limits dialog, double-click on the Minimum, or Maximum, value, as required, and enter a new value. The Minimum value is the minimum number of elements or isotopes that the calculated formula must contain; e.g. if the From value for Cl is 2, the formula must contain Cl2, but can contain any number above this, e.g. Cl3, Cl4, etc. The value is the maximum number of elements or isotopes that the calculated formula must contain; e.g. if the To value for Cl is 2 then the formula must contain Cl2, but can contain any number below this. If the Minimum and Maximum values are the same then the calculated formula must contain this exact number of elements or isotopes; e.g. if the Minimum and Maximum values for Cl are both 2, then the formula must contain Cl2. 2. Select the OK button on each dialog until the display returns to the Elemental Composition Window. Performing a Calibration Calibration can be performed from the Spectrum Window. More: To Make a New Calibration To Apply a Calibration To Modify a Calibration Lock Mass To Make a New Calibration 1. Select the Spectrum Menu Bar Tools, Make Calibration command; the Make new calibration dialog is invoked. 2. Select a Reference file from the dropdown list box. 3. Select the Air references option to include air peaks at 28 and 32 in the calibration. 4. Select the Mass Measure button; the Mass Measure dialog is invoked. Enter the required parameters. For more information see The Mass Measure Process. 5. Select OK. When processing is complete, the Calibration Report Window will be displayed. 6. Parameters can be changed by selecting the Menu Bar Edit, Calibration Parameters command; this invokes the Calibration Parameters dialog. 7. Change the required parameters and select the OK button to display the updated Calibration Report. 8. Select the Calibration Report Window OK button to accept the calibration. A dialog informing of a successful calibration is displayed. 9. Select the OK button. To Apply a Calibration 1. Select the Spectrum Menu Bar Tools, Apply Calibration command; the Apply Calibration dialog is invoked. 2. Select the OK button to apply the calibration. A dialog informing of a successful recalibration is displayed. 3. Select the OK button. To Modify a Calibration 1. Select the Spectrum Menu Bar Tools, Modify Calibration command. The Modify Calibration dialog is invoked. 2. Change the Gain and/or Offset. 3. Select the OK button to apply the modification. Lock Mass The Lock Mass feature allows the User to specify a mass that will be located in the spectrum and used to calculate an offset that can be applied to the rest of the spectrum. The Lock Mass dialog is invoked by the Menu Bar Tools, Lock Mass command. Lock Mass The mass nearest to that specified in this text box [i.e. within (Da/e) the limits specified in Window (Da/e)] will be located in the spectrum and used to calculate an offset that can be applied to the rest of the spectrum. Window (Da/e) Specifies the degree of error allowed when locating the Lock Mass. Use When selected, the lock mass is matched to the nearest Monoisotopic monoisotopic peak in the spectrum. Peak (singly-charge d only) Copying to and from the Windows Clipboard The Windows Clipboard can be used to move data into, or out of, the Spectrum window, either as a picture, or as a text list. For example, spectra or chromatograms can be pasted into reports written with a Windows compatible word processor. The Spectrum Menu Bar Edit, Copy Picture command copies both a metafile and a bitmap to the Clipboard. When the metafile is pasted into another Windows application, it can be re-scaled, if required, without distorting the original image, as long as the original aspect ratio is maintained. More: To Copy a Spectrum as a Picture to the Clipboard To Copy a Spectrum as a Text List to the Clipboard To Paste Information from the Windows Clipboard into a Spectrum Window Removing Pasted Input from the Display To Copy a Spectrum as a Picture to the Clipboard 1. Produce the required display in a Spectrum window. 2. Select the Tool Bar button, or select the Spectrum Menu Bar Edit, Copy Picture command. The contents of the window are copied to the Clipboard as both a metafile and a bitmap. 3. To read the image into another application as a metafile, select the application's Edit, Paste command. Alternatively, select the application's Edit, Paste Special command to have the option of pasting either the metafile or the bitmap image. To Copy a Spectrum as a Text List to the Clipboard 1. Display the required mass range in a Spectrum window. 2. Select the Tool Bar button, or select the Spectrum Menu Bar Edit, Copy Spectrum List command. The displayed section of the spectrum will be copied to the Clipboard as (mass, intensity) pairs. 3. To read the information into another application, select the application's Edit, Paste command. To Paste Information from the Windows Clipboard into a Spectrum Window 1. Select the Tool Bar button, or select the Spectrum Menu Bar Edit, Paste command to paste the default Clipboard object to Spectrum. Choose the Edit, Paste Special command to choose which object to paste into the Spectrum. These objects would typically be metafiles, bitmaps, or text. 2. Use the mouse to drag the outline of the image to the required position. Any contents of the Clipboard, be it a bitmap, a metafile or text, can be pasted into a Spectrum window. If the data is in textual or metafile form, it can be re-scaled using the mouse, and there will be no distortion of the image. However, if a bitmap is pasted, re-scaling is done by stretching the image; this will cause some distortion. To avoid this, scale the image to the required size before copying it to the Clipboard. Removing Pasted Input from the Display 1. Click on item to be removed. 2. Press the keyboard Delete key. Manipulating Library Spectra To Display a Library Entry To Append the Current Spectrum to the Current Library To Display a Library Entry 1. Select the Spectrum Menu Bar Edit, Library, Get Spectrum command. The Display Library Spectrum dialog is invoked. 2. If required, select a new library by selecting the File button. The Open Browser is invoked; select the required library file and select the OK button. 3. Specify an entry number in the Display Library Spectrum dialog Entry: text box. 4. The library spectrum may be added to the current spectrum window, replace the current spectrum, or be placed in a new window. Select the Add trace, Replace trace or New window option as appropriate. 5. Select the OK button. Once a spectrum from a library has been displayed, the rest of the library may be browsed by using the Spectrum Tool Bar button. To Append the Current Spectrum to the Current Library 1. Select the Spectrum Menu Bar Edit, Library, Append command. The Append Spectrum dialog is invoked. 2. If required, select a new library by selecting the File button. The Append File Select Browser is invoked; select the required library file and select the OK button. 3. Select the OK button.