No transition ions found as3e15  2018-08-13 05:52

Dear Skyline team,

I am doing an MRM experiment and am having trouble with one of my peptides.

I have created a spectral library from my samples and used it to identify peptides and transitions. I have ran a dilution series to get a calibration curve and its has worked on all peptides except one. it seems skyline cant find any transition ions for a specific peptide.

If it was just in my heavy i would think that perhaps there is just no transitions present but there isn't any in my light either. the light is the same sample i used to create my spectral library so i would expect to see it.

i have the transitions settings set to targeted acquisition. i have product ion selection = from ion 1 To last ion (i thought this would help but it didn't)

why cant skyline find the transitions?

i have attached a screenshot to help.

Thank you in advance,


Nick Shulman responded:  2018-08-13 13:15
The usual reason that you would end up with no MS2 chromatograms in a PRM experiment is that Skyline did not find any MS2 scans that isolated an m/z which was close enough to your peptide.

The "Method match tolerance m/z" specifies how close the isolation m/z in a PRM experiment has to match your peptide. This setting is on "Settings > Transition Settings > Instrument" and has a default value of .055.

The other thing that often happens in PRM experiments is that there is some other peptide in the document whose m/z is a better match for the MS2 scan than the peptide that you hoped it would match. If MS/MS Acquisition Method is "Targeted", then Skyline will match any particular scan to only one peptide in your document. If you were hoping that your scans would be able to do some sort of double duty, and be applied to all peptides within some small m/z range, then you should change the Acquisition Method to "DIA", and perhaps use the isolation scheme called "Results Only".
It does not look like this is your problem, since you only have one peptide in your whole document. However, if you had other peptides with really close m/z's that you recently deleted from your document, you might want to try doing:
Edit > Manage Results > Reimport
and see whether this problematic peptide gets its MS2 chromatograms after you have removed all of the other peptides from your document.

If you send us your files, we can probably tell you what is going wrong.

In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that .zip file is less than 50MB, then you can attach it to this support request. Otherwise, you can upload it here:

You should also send us at least one of your raw files.
-- Nick
as3e15 responded:  2018-08-14 01:50
Dear Nick,

my method match tolerance m/z is set relatively high at 0.25 so i would hope this isn't the problem otherwise it would imply a large shift. i have run these samples 3 times now all with the same results, but have ran other samples in the interim and they show up well in skyline (just to rule out instrument malfunction)

I know it is not the most efficient way but i have a separate skyline file for each of my peptides because its easier to handle and as you say, i can select targeted acquisition and not worry about mis-matching ions to the wrong peptides.

The skyline share file is small enough to send through here but the Raw data is not so i will use the link you provided.

Thank you for the help.

Nick Shulman responded:  2018-08-14 03:33
Thank you for sending those files.

It looks like you need to set the method match tolerance a little higher.
In your Waters raw file, there are MS2 scans with the precursors 915.75 and 919.759.
In your Skyline document, the m/z of your peptide precursors are 915.4858 and 919.4929.

Your method match tolerance is currently 0.25. You just need to make that a little bigger (e.g. 0.30) and then when you reimport your results you will see MS2 chromatograms for that peptide.
as3e15 responded:  2018-08-14 06:16
Hi Nick,

Ah, that worked! thank you. Skyline identifies the transitions now but it seems there is very low amounts in my runs.

I know the most probably answer is that there is just a limited abundance of these ions in my data, but could there be any other reason (perhaps in the skyline settings) that is causing this. the precursors are present at high amounts so i would expect at least minimal amounts of transitions. it seems however that most (if not all) are given a "red" score and are at extremely low amounts (if present at all).