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 ProMass 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.
Configuration on the Spectrum Enhancement tab is optional
if you want to configure spectral
background subtraction. The other options regarding library searching
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. If your data file contains only full scan
data, you can leave the filter box blank and ProMass will automatically select
the full scan data type. To use a generic scan filter one of the following
is suggested, depending on your data type:
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
Method feature 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
Method feature or by following the
instructions below:
A properly configured ProMass processing method is shown in the screen shot 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 on the Identification tab of the
processing method. If you want to limit processing to a particular
chromatographic time window change the values in the selected retention
time window box, also on the Identification tab of the
processing method editor.
You also have the
option of limiting selected peaks by Area
% or MS intensity in the ZNova
parameter file. These options are set on the
Reporting tab of the Build ZNova Params parameter
editor.