Table of Contents
These tips relate to principally to Data Independent Acquisition (DIA) method of mass spectrometry.
(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:
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.
(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: http://proteowizard.sourceforge.net/download.html
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.
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).
Powerpoint slides with useful information on the fundamentals of Data Independent Acquisition (DIA) are provided below.
Please note that these slide decks are incomplete because slides containing unpublished data have been removed.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: firstname.lastname@example.org
Additional material on DIA:
The attached mini-tutorials explain how to set up DIA methods for the Thermo Q Exactive instrument: