What I mean is that using Skyline to extract chromatograms for both light and heavy peptides in DIA expt with SILAC labelling.

Hi William,

Yes, this is certainly supported. You will want to have a look at our tutorials, including: https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_dia
https://skyline.gs.washington.edu/labkey/wiki/home/software/Skyline/page.view?name=tutorial_existing_quant

and also if you search the Skyline support board for "DIA SILAC" you will probably turn up some useful insights from others who have had similar questions.

Thanks for using the Skyline support board!

Brian

Thank you, Brian.
I have read the two tutorials. However, I have same question to the previous user asked in support board which is unanswered (topic "SWATH quantitation" by song.li@uconn.edu). How can Skyline find heavy DIA data for proteins with no heavy data in the library? There seemed to be contradictive opinion about whether to used labelled approach in DIA experiment, I appreciate your view on it.
Our planned workflow is that first generate spectral library by running DDA (fractionated samples with iRT standands spiked in) and spike SILAC internal standard to samples for DIA experiment for better quantitation performance. Is there any caveats for it?

William

Hi William,
If you think about DIA as a targeted experiment there is no difference between PRM and DIA. You can list peptides in the tree with or without stable isotope labels and extract and integrate the chromatographic peak areas. Skyline can make use of a spectrum library and iRT values to determine the best retention time to use as a measure of the target peptide. Webinars 14 and 15 describe how to use a spectrum library to analyze DIA data.

SILAC or any other stable isotope labeling method can be used as a way to normalize the signal intensity. It is definitely compatible with DIA.

The LINCS project is performing DIA on phosphopeptide enriched samples and are analyzing ~100 phosphopeptides with stable isotope labeled internal standards. https://panoramaweb.org/wiki/LINCS/Overview%20Information/page.view?name=LINCS%20PCCSE%20Overview

-Mike

Just avoid targeting b-ions in general, since those will be the same for light and heavy if both fall within the same DIA isolation window. This is the only difference I can think of between SILAC for DIA versus PRM and SRM.

Thanks for posting to the support board.

--Brendan