DIA 3-plex SILAC trinidad martincampos  2020-09-27

Hello all,

First of all, thank you very much for the effort you put into Skyline for all of us ;)

I took a look at the post with "DIA SILAC" but my problem is still not solved... I have a 3-plex SILAC (light, medium and heavy) experiment that I am going to analyse with DIA in an orbitrap fusion.
Any hint about how could I do that? What happens with a peptide whose light and medium (for example)tags are in different windows? Any possible workflow to do this?

Thank you very much!!!

Nick Shulman responded:  2020-09-27
I do not believe it is a problem if your light and heavy precursors end up in different isolation windows. Any MS2 scan whose isolation window includes the monoisotopic m/z of the precursor will contribute a point to the chromatograms for that precursor. The chromatograms for your two precursors might come from slightly different MS2 scans, but that will not necessarily prevent them from being able to be compared to each other.

I believe a bigger problem is that your DIA windows might cover different amounts of each precursor's isotope envelope. For this reason, in order to compare the light and heavy MS2 intensities, you would need to correct for the fact that a different amount of each precursor fit in the isolation window.

One way that you could work around this problem would be to collect your data using staggered (overlapping) DIA windows. Each precursor would be isolated in multiple sets of DIA windows. Then, you could define an isolation scheme in Skyline (at Settings > Transition Settings > Full Scan > Isolation Scheme) which specified that the DIA windows had wide margins. In this way, Skyline would only use MS2 scans where the monoisotopic mass of the precursor was not close to the edge of the isolation window, so that all of your chromatogram intensities would be comparable to each other.
(I am not aware of anyone who has actually done this overlapping isolation windows with wide margin technique to work around partial isotope envelopes being isolated, so I do not know whether it works in practice).

The usual thing that people do is to not use DIA, and instead do PRM with MSX where you tell the mass spectrometer to isolate more than one of your peptide's precursors in a single scan.

Hope this helps,
-- Nick