The ability to input sequences of
biomolecules in the Xcalibur sample list for automatic calculation of target masses ProMass is particularly useful for confirming target masses of oligonucleotides and proteins. In this example, we'll demonstrate the processing of oligonucleotide mass spectra using ProMass. This example will highlight several important features of ProMass including:
The ability to input sequences of
biomolecules in the Xcalibur sample list for automatic calculation of target masses
The ProMass browser and the color-coded display
The sample-plate viewer
A note about data acquisition for this example:
In this example, we used the negative ion mode to analyze the oligonucleotides. We set up our instrument and autosampler to perform a rapid on-line desalting step, which gave us a single chromatographic peak for each data file. The Xcalibur processing method (oligo.pmd) was set up to process the single biggest chromtographic peak in the full scan, negative ion, centroid mode.
Locating the oligo example files:
The oligo example files are located in your install directory under TestData\oligos. This example assumes that you have installed ProMass in the default installation directory, C:\Program Files\ProMassXcali. From the Xcalibur Sequence Setup view, open the sequence file oligo.sld, which is located in the oligo example directory (e.g., C:\Program Files\ProMassXcali\TestData\oligos\oligo.sld). You should see a sequence list of 5 data files oligo01 - oligo05. The sequence list specifies a processing method, a Znova parameter string, and a BioSequence string for each data file, as shown below:
Have a look at the sample list and note the information that is entered in the various fields, e.g., Target Info, BioSequence, Znova Params, and Proc Method fields.
Note: If you did not install ProMass in the default install directory, you will need to update all references of C:\Program Files\ProMassXcali in the sequence list to your install directory. Update the fields in the Path, Proc Method, and Znova Params if this is the case. Also, use the Check Processing Method feature to ensure that the oligo.pmd processing method has the correct information about the location of the ProMass program.
Processing the data:
To process the data, select the
sequence rows with your mouse and click the Batch Reprocess button 
on the Xcalibur Sequence Setup toolbar. The oligo example
files should begin processing. The Xcalibur processing queue will beep when
processing is finished.
Viewing the results:
If the results from the ProMass processing are not already displayed in an Internet Explorer window, go to the ProMass Home Page and click the ProMass Browser button. The results should look something like the screenshot shown below:
Explore the results by clicking on the color-coded sample wells or by the clicking the links in the table to access the detailed summary for each sample. If you have Excel installed on your system, an Excel summary should also have been produced and should still be open after processing. Click on the RESULTS sheet to examine the Excel summary for these samples.
A review of the ZNova Parameter file for this example:
A number of the ProMass features have been enabled via the oligo.params ZNova parameter file used in this example. From the ProMass Home Page, click the Build Params button and open the parameter file used in this example (e.g., C:\Program Files\ProMassXcali\TestData\oligos\oligo.params). On the Reporting tab note that the Show Sample Plate and Use Excel Summary options have been checked. These enable the 96-well view and the Excel summary, respectively. Also note on the Advanced Deconvolution tab that the output mass range has been left blank. This enables ProMass to automatically deconvolute a +/- 25% mass window around the expected target masses.