Table of Contents

   DIA (Data Independent Acquisition)
     Generating an Overlapping Window Isolation List using Skyline
     Full Spectrum Demultiplexing of Overlapped DIA Windows using MSConvert
     Slides Explaining Data Independent Acquisition
     DIA Methods for Thermo Q Exactive

DIA (Data Independent Acquisition)

Generating an Overlapping Window Isolation List using Skyline

(This tip relates to Skyline v 4.2 or later.)

This is a quick demonstration on how to use Skyline to generate an overlapping-window isolation window list suitable for acquisition using the approach described in this manuscript and downstream computational demultiplexing.

To begin this demo, start with a blank Skyline document: 

  • From the Skyline landing page, click Blank Document

  • On the Settings menu, click Transition Settings and in the Transition Settings dialog box, click the Full Scan tab.


  • Under MS/MS filtering, click the Acquisition method dropdown and select DIA. 
  • For the Isolation scheme, select Add ...


  • In the Edit Isolation Scheme dialog, enter a name for the Isolation scheme such as '20mz_overlap' and then click the Prespecified isolation windows radio button.  


  • Click the Calculate ... option and enter parameters for the method.  
  • Enter '500' for Start m/z.
  • End m/z is '500'.
  • Window width should be '20'.
  • Be sure that Deconvolution is set to Operlap and that Optimize Window Placement is checked.


  • Click OK to close the Calculate Isolation Scheme dialog and OK to close the Edit Islation Scheme window.
  • Click OK once again to close the Transition Settings dialog.
  • From the main Skyline menu, select File and then Export.  From the flyout menu, click the Isolation List ... option. 


  • In the Export Isolation List window, select the appropriate instrument (for this example, 'Thermo Q Exactive') and choose a file location in which to save the list of isolation windows.


The resulting isolation list contains the isolation centers of each isolation window in the order that they should be acquired. NOTE: the isolation centers will need to be regenerated using this approach if any of the other acquisition parameters such as isolation width or m/z range covered are changed.

Full Spectrum Demultiplexing of Overlapped DIA Windows using MSConvert

(This tip relates to Skyline 4.2 and later.)

This is a quick demonstration on how to use MSConvert to generate a demultiplexed dataset from an input datafile(s) containing spectra with overlapping data independent acquisition windows. In the case of the overlapped window approach described in this manuscript, the output from MSConvert will contain twice as many spectra as the input (two demultiplexed spectra are generated from each acquired MS/MS spectrum). This tutorial uses MSConvert distributed with ProteoWizard version 3.0.18328 with vendor libraries downloadable here:


  • Open MSConvert and select the Browse option to select the input overlap-multiplexed files.  
  • Select the SIM as spectra option.



  • OPTIONAL: Change the output format for the file using the Output format drop-down menu. This tutorial outputs mzML.
  • Add a Peak Picking filter to the data by selecting the options indicated below and clicking the Add button below the Filters section:


This causes the MS2 data to be centroided prior to demultiplexing, which is currently a requirement for full-spectrum demultiplexing using MSConvert. If the data were acquired with centroiding enabled, this step will have no effect and demultiplexing will proceed as expected.


  • In the Filters section, add a Demultiplex filter with optimization set to Overlap Only  with the settings shown below. 
  • Click the Add button.



Note that the mass error may need to be adjusted depending on instrument platform. The mass error should be set to the maximum error expected in m/z measurement of the same analyte in subsequent spectra. Note that this measurement is of expected deviation of a measurement from spectrum to spectrum, not its deviation from the correct theoretical m/z (mass accuracy).

  • Click Start to output demultiplexed files.

Slides Explaining Data Independent Acquisition

Powerpoint slides with useful information on the fundamentals of Data Independent Acquisition (DIA) are provided below.

Introduction to Data Independent Acquisition

DIA Method Design

DIA Data Analysis

Future Directions of DIA

Please note that these slide decks are incomplete because slides containing unpublished data have been removed.  If you would like the full slide deck for your personal use, or would like to use some of these slides in your own presentation, please contact the MacCoss Lab at:

Additional material on DIA:

DIA Tutorial

DIA Methods for Thermo Q Exactive

DIA Webinar

DIA Methods for Thermo Q Exactive

The attached mini-tutorials explain how to set up DIA methods for the Thermo Q Exactive instrument: