You haven't actually uploaded any files.

If they are less than 50mb you can attach them here. Otherwise you can upload them here:

https://skyline.ms/files.url

Nick,
yes - got error when submitted message. Now I am getting error on the files upload url that I do not have privileges to upload there.
Attaching the Skyline zip here.

Tomas

Never mind, it seems like it uploaded despite the error message.

Tomas

Thanks for sending those files.
I am not sure what I am supposed to be seeing. The chromatograms look fine to me, but I am not sure what I am supposed to be looking at.

Can you attach a picture of what you are seeing that looks wrong?

Problem at least on my Skyline is with the peptide SVSLPSLDPASAK in sample A10c.
Attached is screenshot from Skyline and from Xcalibur FreeStyle. Skyline gives me mess - but the precursor is there when I look at it in FreeStyle and will also show up in the MSMS with expected fragments ions.

 Tomas

I am not sure how to interpret the display of Thermo FreeStyle, but it looks like you are looking at the MS1 chromatogram for the m/z 640.35.

Your Skyline document only has MS2 transitions in it, which you would not expect to look like a precursor chromatogram.

If you want to add precursor chromatograms to your Skyline document, then you can do the following:
1. In
Settings > Transition Settings > Filter
add "p" to the list of ion types.
2. In
Settings > Transition Settings > Full Scan
under "MS1 filtering isotope peaks included" change it from "None" to "Count".

Then, if you reimport your results, you will get some precursor chromatograms.

In the Skyline document that you sent me, Skyline chose the peak at 23.4 minutes as the best peak. This was done based on looking at all 4 precursors under that peptide (heavy/light & charge 2/3). Only heavy charge 2 has any real signal at that time.

What do you think Skyline is doing incorrectly?

I am sorry Nick, I am not very good at explaining the problem. What is shown in the FreeStyle is MS1 of the single synthetic peptide in the sample. It elutes at 24.6 min as indicated by the MS1 (it is hard to do in QualBrowser or FreeStyle what you do in Skyline), but basically if I take any of the expected fragment ions in FreeStyle and make its extracted ion chromatogram in FreeStyle using the MS2 data for the corresponding 24 m/z window of the DIA data I will get exactly what the MS1 data shows - a nice strong peak at 24.6 min.
The problem is that the upload into Skyline does not reproduce that. When uploaded into Skyline you get the messy trace with some sort of a peak at 23.4 min, which is not the correct peak - you would expect to see exactly what I see with FreeStyle. I have now added a slide with XIC of the y9 ion m/z 893.48 (this peptide is heavy labeled - the light precursors are there just for check of isotopic enrichment). Now you can see that when you export this fragment ion you get the same peak as in MS1 at rt 24.6 min. And that is exactly what you should see in Skyline, but you do not. Instead you see nondistinct peak at 23.4 min.

I hope this makes it clearer what I am trying to convey.

Tomas

I believe that your graph from FreeStyle is showing a precursor 652.5760. I assume that means it's looking at the scans that isolated 640.576-664.576.

In Skyline, that peptide has a precursor m/z of 640.3528. Skyline is only going to be looking at the MS2 scans that isolated 616.564-640.564.
When Skyline is deciding which isolation window to use for a peptide, Skyline only looks at the monoisotopic mass of the peptide.

It sounds like you want Skyline to use the next higher window.

I am not sure what we recommend that people do in this case.
You could redesign your method so that your isolation windows have more overlap, and you could avoid cutting off too much of your isotope envelope.

Does anyone else have any ideas?

I do not quite understand this, but when I look at the MSMS of the lower DIA window (center 428.56, width 24) where the precursor m/z 640.35 should be (628.56+12=640.56, the fragment ions appear to be 1 amu lower i.e 892.47 instead of 493.47, and the fragments show correctly in the DIA window (center 652.5768), where 640.35 should not be (652.58-12=640.58). I guess issue of fringe effect where the selection window for MSMS is not exactly square and may be shifted slightly towards lower m/z.
So I guess this is not problem of Skyline, but the fact that the precursor fell exactly on the border between two DIA windows.
If somebody has suggestions I would appreciate it.

Thanks Nick.
Tomas