Application Example: Intact Protein LCMS

This example will use one of the data files included with the ProMass program to show you how to automatically process intact protein LC/MS data.  This example will highlight the following features:

 Specifying and reporting multiple target masses

 Setting up a processing method to select multiple peaks from a chromatogram for deconvolution

 Viewing ProMass results from LCMS data in the ProMass browser

If you have not already done so, please review the Getting Started... topic on automatic processing, as this will instruct you on the basic procedures involved in automatic processing.

A note about the data in this example:

This example shows the LCMS separation of a protein mixture containing 5 expected proteins: insulin (M=5733.5), RNAse A (M= 13682.2), RNAse B (M= 14178.0), lysozme (M= 14305.1), andcarbonic anhydrase (M= 29022.6). 

Locating the protein LCMS example file:

The protein LCMS example file is located in your install directory under TestData\protein lcms. 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 protein_lcms.sld, which is located in the protein lcms example directory (e.g., C:\Program Files\ProMassXcali\TestData\protein lcms\protein_lcms.sld). You should see a sequence list with a single data file named 'protein_lcms'. The sequence list specifies a processing method, a ZNova parameter string, and a Target Info string, as shown below in the two screen captures.  You will have to scroll horizontally to view all of the sample list fields.

Note the following:

• A processing method is defined in the Proc Meth field, which determines how peaks in the chromatogram are selected for deconvolution.
• A ProMass parameter file is specified in the ZNova Params field, which specifies the options for deconvolution.  Note how the parameter file is referenced with the -P syntax followed by a quoted string for the parameter file name.
• Multiple target masses have been specified in the Target Info field as comma separated values.  These masses represent the expected masses of all 5 of our target proteins.

Examining the Processing Method:

• Right-click the entry in the Proc Meth field in the sample list to select the protein_top_2% processing method file and open it in the Processing Setup program.
• After the Processing Setup program opens, click on the Qual button in the left panel.  The display should look as shown below:

• The gray shaded areas of the chromatogram show which peaks will be selected for deconvolution.  The limits of each peak serve as the start and end scans that will be collected into a single averaged scan at each retention time for ProMass deconvolution.  The red rectangle in the chromatogram view illustrates the time range that will be used for peak detection.  Also note that the chromatogram threshold has been set to the highest 2% of detected peaks.  Experiment with the settings in this window and observe the effects on the detected peaks in the chromatogram view.  You will have to click the OK button to see the effects of your changes.
• You can use this processing method as a template for protein LCMS data that you have aquired in your laboratory.  You can test this processing method on your own data file by opening your raw data file in the processing method.  To do this, select Open Raw File... from the File menu in the Processing Setup program.  Adjust the settings to get the desired peak selection results.  Do not overwrite this processing method, as it will be used to demonstrate the processing example for this exercise.   A separate help topic is also available for processing method creation .
• Exit the Processing Setup program when you are finished experimenting.

Processing the data:

• Return to the Xcalibur Sequence Setup view and select Batch Reprocess... from the Actions menu of the sample list, or simply click the  button on the tool bar.  The Batch Reprocess dialog box will be displayed.
• Ensure that the following options are set: Qual - Peak Detection & Integration, Programs, and Replace Sample Info.  The dialog should look as shown below.
• Click on OK

• When processing begins, the ProMass console window will pop up or will be displayed in the task bar.  The console will automatically dismiss when ProMass processing is complete.

Viewing the results:

• When processing is completed, use the ProMass browser from the ProMass Homepage to locate the newly processed data and display it in your web browser.
• Click on the data file name in the ProMass report in your web browser to display the detailed report page for the protein LCMS data.  The report should like the screen capture shown below.
• Experiment with the navigation links in the report to view ESI and deconvoluted spectra associated with each chromatographic peak.