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What is ProMass?

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How to run ProMass

The steps for running ProMass in a fully automated mode are shown below.  If you want to manually process spectra, refer to the section on processing spectra from the clipboard.

• Create and save an Xcalibur sample sequence
• Define a Qual Processing method for picking qualitative chromatographic peaks
• Check your processing method for the proper format
• Build a ZNova Parameter File
• Add ZNova Parameters to your Xcalibur Sequence
• Run or Batch Reprocess your Xcalibur Sequence
• View Results

Refer to the Xcalibur documentation on the basics of how to create and save sequences. If you are using ProMass for the first time you will probably need to modify the sequence column arrangement to include fields needed to run ProMass. If you have completed the Getting Started Example, you can skip this step and go straight to defining a qual processing method.  Xcalibur does not know about the special fields required by ProMass.  The easiest way to have Xcalibur recognize these fields is to use the ZNova-ize Sequence feature from the ProMass home page.  Click on the ZNova-ize Sequence button and open one of your existing Xcalibur sequences.  Click on the Save As button and save your sequence to a new file name. Click on the Done button (the green check mark) to exit from the ZNova-ize Sequence program, and click on the File | Exit menu item to exit ProMass.  Now, go back to the Xcalibur Sequence Setup program. First, open the sequence that you just saved in ProMass. Access the Change...|Column Arrangement... dialogue in the Xcalibur Sequence Setup window. You should see the following dialogue:

Make sure to select and add the Target Info, BioSequence, and ZNova Params columns to the displayed columns.  You only need the Target Info, and BioSequence fields if you plan on entering peptide or oligonucleotide sequences into your Xcalibur sample list.

Refer to the Xcalibur documentation for the details of how to create processing methods. Basically, the processing method tells Xcalibur how to process chromatographic peaks. When a data file is processed through ProMass, an Xcalibur results (.RST) file is always created which indicates how the peaks were defined during processing. ProMass uses the results file to determine which scans to average as input to ZNova deconvolution. You setup a Qual processing method by first accessing Processing Setup from the Xcalibur home page. An example is shown below:

The example shown is a very simple processing method that picks the single biggest TIC peak in the chromatogram. The grey shaded area of the chromatogram shows what will be considered as the start and end scans in ZNova processing. The following items are required for proper ProMass execution:

• Always make sure you select a Qual processing method
• A scan filter which indicates a full scan data type must be specified for ProMass to operate properly on a data file. To use a generic scan filter one of the following is suggested, depending on your data type:
  • + p ESI full ms (positive ion profile mode full scan ESI)
  • - p ESI full ms (negative ion profile mode full scan ESI)
  • + c ESI full ms (positive ion centroid mode full scan ESI)
  • - c ESI full ms (negative ion centroid mode full scan ESI)
• You can pick peaks based on the TIC, the Base Peak trace, or a Mass Range trace.
• The ProMass executable must be installed with each processing method intended for ProMass processing. The Check Processing Method feature will perform this step for you, but we will describe here how to perform this step manually. In order to do this select the Programs button on the left side of the Xcalibur Processing Setup. A properly configured ProMass processing method is shown in the screenshot below. Ensure that the following are set: Enable = Yes, Action = Run Program, Program or Macro Name = promassxcali.exe in your install directory, Sync = Yes, Parameters = %S %R %F

• The best way to see how your data will be processed is to open a raw file in the processing method and check the peak picking. To do this, in the Processing Setup File menu select Open Raw File...
• It is suggested that you use ICIS peak detection, as it is often easier to define the limits of peaks. If you want to use only the tops of the peaks, use a low baseline window value (in the ICIS Peak Integration box). If you want the scans to be averaged near the baseline, increase the baseline window value.
• If your data have a lot of peaks, you may want to limit the number of peaks by selecting one of the Limit Peaks options or selecting a relative peak height threshold. If you want to limit processing to a particular chromatographic time window change the values in Range box.

The easiest way to add ProMass processing to an existing processing method and to ensure that the programs and scan filters are configured properly is to use the Check Processing Method feature on the ProMass home page. Launch the ProMass home page and click the Check Processing Method button.

• Open your processing method
• If you see the following dialogue, it indicates that the programs section of your processing method is incorrect.

• Click on OK.

• Click Yes to fix programs section.
• If your scan filters are not configured properly you will see the following dialogue:

• Click on OK.

• Click Yes to fix the scan filter.
• Select your the ion mode polarity (Yes = positive; No = negative):

• Select your data type ( Yes = profile mode; No = centroid mode)

• Once you have finished updating your processing method, you may want to check it again to make sure everything got set properly.  Click the Check Processing Method button.  You should see the following dialogue if your processing method is ok.

Build a ZNova Parameter File

ZNova parameters are used to instruct ZNova how to deconvolute your spectra and where to put the results.  Here we will provide some basic information about how to build and save parameter files.  For more detailed information about what each parmeter does, refer to the ZNova Parameters Reference page.  Click on the Build ZNova Params button on the ProMass home page.

You should see a blank ZNova parameters screen.  If you move your mouse over the tool bar buttons at the top you should see help text that identifies the function of each button.  If you move your mouse over any of the parameters areas, information about that parameter will appear in the status area at the bottom of Build ZNova Params window.  From the tool bar at the top, click on the Default Parameters button (the button with the forward and reverse green arrows).  This will restore typical default parameters.  As shown in the dialogue below, select a molecule type for the type of analysis you are performing, then click on OK.

Make sure the following are set:

• If you are deconvoluting a positive ion data file, make sure the Adduct Ion is set to 1.0079 (the mass of a proton), or -1.0079 for a negative ion.
• Select the input deconvolution range, First m/z and Last m/z.  Leave the fields blank to allow ZNova to automatically determine the input range from the scan limits.
• Select the ouput deconvolution range, Minimum Mass and Maximum Mass.  Make sure these settings cover a range where you expect to see deconvoluted signals.
• Set the Directory Format option to raw, so that your processed results will be saved in a subdirectory called promass_results in your data file path.  (Note: the Results Directory setting will have no effect if raw Directory Format is selected, unless the data file path cannot be determined at run time.)

Click on the Save As button on the tool bar to save your parameter file as name that you specify.  Click on the Done button to exit the Build ZNova Params program.  For more information on the ZNova parameters, refer to the ZNova Parameter Reference page.  Refer to the Parameter File Examples Page for additional hints on selecting parameters for various applications.

Add ZNova Parameters to your Xcalibur Sequence

The ZNova-ize Sequence utility is used to add ZNova parameters to your Xcalibur sample list. The parameters determine how the data will be deconvoluted and where the results will be stored.  Move your mouse over each of the toolbar buttons to examine the function of each button.  The basic steps involved in ZNova-izing a sample sequence are as follows:

• Open an existing Xcalibur sequence
• Select the files in the Xcalibur sequence that you want to ZNova-ize by either checking the box next to each desired file name or using the Select All Samples tool bar button.
• Most of the time you will probably want to just add a ZNova parameter file to a sequence.  To select a parameter file, click on the button to the right of the parameter file box at the lower right part of the screen.
• To add parameter strings to the selected samples in the sequence enter a value for one or more parameters and click on the Add Parameters to Sequence toolbar button (the left pointing finger icon).
• Once you are satisfied with your parameter string selections, don't forget to Save or Save As... your Xcalibur sequence.
• Return to the Xcalibur Sequence Setup screen and open the Xcalibur sequence that you just ZNova-ized.
• Your added parameter strings should appear in the ZNova Params section of the Xcalibur sequence.  An example of the strings we added above are shown below.
• Note that any parameter strings that you add in addition to the parameter file will override those same options in the selected parameter file.

Run or Batch Reprocess your Xcalibur Sequence

You batch reprocess a sequence if you have already acquired the data and want to process it post-acquisition.  Use the mouse to select the rows in the sequence that you want to process.  Click on the Batch Reprocess button in the Xcalibur Sequence Setup or access this function from the menu under Actions | Batch Reprocess...  You should see the dialogue box shown below.  Make sure that the same options checked below are checked in your dialogue.  The rows that you selected should appear in the Process Rows box. 

Make sure that only the following options are checked:

• Qual - Peak detection and integration
• Programs
• Replace Sample Info

Click on OK.  If processing proceeds correctly, then a console window will pop-up similar to the one shown below.  This indicates that the Xcalibur processing method has found chromatographic peaks and ProMass has passed spectra to ZNova for deconvolution.

Do not dismiss the console while it is running, or processing of that data file will terminate.  When the entire sequence is finished, the Xcalibur processing que will beep signifying that it is done.

View Results

The easiest way to view your results is to launch the ProMass HomePage and click on the ProMass Browser button (if the button is greyed out, you need to upgrade ProMass to the latest version). 

If you selected the raw Directory Format option in your parameter file, all of your results files will automatically be placed in a directory called promass_results in your data file path.  There are many more options for directing ProMass output.  Review the Directory Format and Results Directory reference if you want to use one of the other options.