Determining Isolation Windows in DIA Raw Files


Scaffold DIA supports loading DIA data that has been captured with one of three types of widows: non-overlapping, overlapping with margins or staggered windows, and multiple instrument types are supported. Non-overlapping or margined windows can either be fixed or variable width. However, only fixed width staggered windows are supported. It is important that the isolation window type is known in order to set the data acquisition type parameter when loading data in Scaffold DIA. 

There are a few programs that allow for visual representation of windowing schemes including Skyline and EncyclopeDIA. The following article will guide users on how to determine the isolation scheme found in a raw file.

Using EncyclopeDIA to Determine Isolation Windows

EncyclopeDIA is an open source program that forms the core of Scaffold DIA. To use Encyclopedia to check an isolation window scheme use the following procedure:

  1. Convert your raw file to mzML using the MSConvertGUI program as part of ProteoWizard. No specific filters, such as demultiplexing or peak picking are needed now. If you have installed Scaffold DIA you likely already have ProteoWizard installed. If not, you can find it here.
  2. EnclycopeDIA can be downloaded here, follow installation instructions provided. Note, the program requires Java to be installed.
  3. Use the View dropdown menu to open the Raw File Browser.
  4. Select your converted mzML using the Edit button found next to the file path.
  5. Click on the Structure tab to see the isolation window scheme. 

Using Skyline to Determine Isolation Windows

Skyline is an open source tool developed by the MacCoss group at the University of Washington. It also can be used to determine isolation windows.

  1. Skyline can read raw files directly, thus there is no need to convert to mzML prior to determining isolation window scheme.
  2. Download the program here and follow instructions for installing.
  3. Open Skyline and select Blank Document
  4. From the Settings dropdown menu select Transition Settings…
  5. Navigate to the Full-Scan tab
  6. Locate the MS/MS filtering section, choose DIA from the Acquisition method dropdown
  7. Click to expand the Isolation Scheme dropdown menu and select Add
  8. Select the Prespecified isolation windows radio button
  9. Click on the Import button to load the raw file in question.
  10. Once the file imports the table will be populated with the start and end values for the windows as well as the margin size (if there are any).
  11. Clicking the Graph button will provide a visual display for the windowing scheme.

Isolation Window Examples


Figure 1. Isolation windows that overlap each duty cycle are considered staggered


Figure 2. Windows that do not overlap per window or per duty cycle are considered non-overlapping.


Figure 3. If each window has a small overlap on each side of it, those are overlapping with margins. Note, the pink shaded areas.

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