DIA data upload with 15N-APOA1 iRT peptides and Chromatogram Library (elib)

DIA data upload with 15N-APOA1 iRT peptides and Chromatogram Library (elib) Tomas Vaisar  2020-04-09 10:13

Brendan et al,

This may be covered somewhere, but I could not find it in any tutorial or here on the Support pages.
I have 15N-APOA1 iRT peptides in my samples and I enumerated proteins/peptides (database search) in my samples using the Chromatogram Library approach (6 DIA runs of the pooled sample with overlayed 4 m/z windows segmented over 400-1000 m/z). I ran the data through EncyclopeDIA (Walnut search part) and created a chromatogram library (elib). Now I want to get this into the Skyline and create iRT calculator using the ChromLibrary data so that I can upload the data from all study samples using the iRT predictor.
Where this fails is that while I can create the iRT calculator for the 15N-APOA1 peptides alone, I cannot find a way to add the remaining peptides to the iRT calculator. The 6 DIA "Chrom Library" files load fine into the Skyline document (using Add one replicate), but whenever I try to update the calculator it fails because the 15NAPOA1 peptides are not present in all Chromatogram Library mzML files. (I am not uploading precursor ions only b,y fragment ions).

What is the way to get this working? I am sure there is a way people do it.



Nick Shulman responded:  2020-04-09 10:36
We do not recommend using iRT with multi-injection DIA because each injection will be missing the ms2 for some of the iRT standards.

In theory you could get this to work if you told Skyline to only look at the ms1 chromatograms for the iRT standards, but I believe that does not work very well since ms1 is not sufficient to reliable find the peptides.

Usually people use EncyclopeDIA to search their wide-window single injection quantification runs, as well as to create the multiple injection chromatogram library. Then, they give Skyline the .elib file from the quantification runs, and Skyline uses the peak boundaries from that .elib file. For this reason, they never need to use iRT with an EncyclopeDIA chromatogram library.

I do not know of anyone who has tried doing what you're doing of only using EncyclopeDIA on the chromatogram library part of the search, and then trying to use Skyline on the quantification runs, and I do not know whether there is a way to make it work well.

-- Nick
Tomas Vaisar responded:  2020-04-09 10:57
Thanks Nick.
Hmm, I am surprised nobody tried this, I wonder why. In theory it seems to me straightforward - you have your Chromatogram Library that should contain all peptides you should be finding in your quantification runs (theoretically, but especially if you do a multiple injection Chromatogram library for each of your study groups) and then you use these runs to create the iRT calibrator with all peptides. Since it is few runs and you are doing a single injection for each peptide (nominally), you probably do not need to get the iRT accurate, but at the end you will have your iRT calculator populated with all peptides you care about, and the you apply this calculator to your quantification runs where they will get readjusted.

I do understand the issue of the iRT in multi-injection runs, but it is only used to get the initial RTs.

Oh well, will have to go different route.

Thanks again,
Nick Shulman responded:  2020-04-09 11:37
I understand what you are asking now. Sometimes you have multiple injections that took place right after each other on the same column on the same mass spectrometer and you are confident that the retention times did not change between the injections.

You can use ProteoWizard msConvert to create a single .mzML which has all of the spectra from all of your injections ("--merge"). Be sure to include the "--filter sortByScanTime" argument as well so that the spectra from all of the files get interleaved in the right way.
This seems like something that would work perfectly, but I might be overlooking something.
-- Nick
Tomas Vaisar responded:  2020-04-09 11:45

Thanks - that sounds like a great option. I will try to combine all the ChromLibrary multi-injection runs into a single mzML file - that way Skyline should be happy about having the 15N-APOA1 iRT peptides there. I've used the Import>Add single replicate, but that's not exactly the same thing as single mzML as it always need the multiple mzML files to "reupload" when adding the other peptides to the iRT calculator.

I will let you know if that works.

Tomas Vaisar responded:  2020-04-10 08:52

Thanks a lot - this works like a charm (well - a slow charm, because combining the 6 mzML multi-injection files takes couple hours on our box, 12 cores/24 GB RAM - not sure if there is a way to make it go faster, and makes huge file - 6GB).
But after that everything works just fine.
Your advice really appreciated.