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 processing method is shown below using our test.pmd method from the Getting Started example. Open the test.pmd method in the TestData directory of the ProMass install directory (e.g., C:\Program Files\ProMassXcali\TestData\test.pmd). Click on the Qual button at the left in the Processing Method Setup window. Open a raw file by selecting the File | Open Raw File… menu option. You can use our myoglobin LC/MS file (myolcmsdata.raw) which is found in the TestData subdirectory.
The example shown is a very simple processing method that picks the single biggest TIC peak in the chromatogram. The gray shaded area of the chromatogram shows what will be considered as the start and end scans that will be collected into a single averaged scan for ZNova deconvolution. The following items are required for proper ProMass execution:
Always make sure you select a Qual processing method. You only need to configure items in the Identification tab. The other options (Spectrum Enhancement, Library Search, etc.) in the Qual Processing Method Setup are not used for ProMass processing.
In the Detector section of the
processing method, select MS as the detector type and
ICIS as the Peak Detect method.
In the Filter box, select a scan filter which indicates a full scan data type. It is suggested that you use a
generic scan filter, for example, one that does not specify a mass range. This
way your processing method will work on a wide variety of data files that may
have different scan limits. 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)
Use the Trace box to select peaks based on the TIC, the Base
Peak trace, or a Mass Range trace. Typically, the TIC trace is used for most applications. However, you may find that Base Peak is better for peptide applications.
ProMass also
supports automatic background subtraction with the Xcalibur Spectrum Enhancement
feature. For information on setting up background subtraction see the help
topic Automatically
Subtracting Background Spectra.
The ProMass executable file must be installed with each processing method in the Programs section in order for ProMass processing
to occur. The Check Processing Methodfeature on the ProMass Home Page will add the programs section for you automatically. If you are creating a processing method from scratch, as opposed to editing an existing ProMass-enabled method, you will need to make sure the Programs section is properly
configured by either using the Check Processing Methodfeature or by following the instructions
below:
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
Here are some additional hints for creating Qual processing methods:
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 parameters. 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 many 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.