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retention time restriction for targeted EIC determination

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retention time restriction for targeted EIC determination Jens  2017-09-28 08:35
 
Dear Skyline-Team

I am currently looking for a software, which is able to semi-automatically extract EICs for certain defined peptide species (e.g. non-deamidated and deamidated peptides and/or non-oxidized and oxidized peptides etc.) only in defined retention time windows, just based on MS1 (Precursor+corresponding isotopes) and without a previous identification e.g. using a search engine. In Skyline (v3.5.0.9319) I was able the create a transition peptide list for different charge states of such peptides and use it to get EICs. But, unfortunately I was not able to define retention time limits for gaining those EICs. When I tried to put in explicit retention times and windows (e.g. in the document grid under peptide view) it was ignored by the software (or I did it wrong?).
The retention time restrictions would be necessary to separately determine EICs for e.g. iso-aspartate and deamidations of a peptide, which have exactly the same mass (and isotopes), but different elution times/windows. The goal would be to get those EICs without correcting retention times or reassigning peaks.
Is it possible to define retention time limits/restrictions within Skyline in which the EIC for a certain species is determined and use it to get EICs directly from Thermo-raw-files?

Thanks a lot in advance!

Best regards,
Jens
 
 
Brendan MacLean responded:  2017-09-29 08:17
Hi Jens,
Can you post a screenshot of your Transition Settings - Full-Scan tab? If Skyline is ignoring the explicit settings you have made, then the first most likely explanation is that you have (accidentally) told it to do that by specifying your extraction retention times as "Include all matching scans", instead of using "Use only scans within [] minutes of predicted RT", which is the only setting where Skyline consults the explicit retention times you have set.

I can see the problem in this case, since you have also set an explicit window and the specified minutes would seem to override that.

Perhaps we should make "Include all matching scans" respect explicit times, as we still allow Transition Settings - Instrument - Min and Max time to restrict the definition of "matching scans".

You are the first to run into this. So, it may be that we have not thought it through thoroughly enough, but I am still interested in knowing whether you could at least get part of what you are looking for by using the "predicted RT" option in the Transition Settings - Full-Scan tab, or if you are already using that, then we'll need to keep searching for why Explicit Retention Time is getting ignored in your document.

--Brendan
 
Jens responded:  2017-10-02 04:50
Hi Brendan,

thanks for your response.
Yes, you are totally correct in assuming that I used the "Include all matching scans" setting. But this was due to the fact that Skyline is asking for a Prediction algorithm when I am using "Use only scans within [] minutes of predicted RT" (see attachment). I then tried to include the list of modified peptides with their RTs in the predictor list, but it is giving me the response, that there are peptides with the same mods (as e.g. deamidation and iso-aspartate result in the same mass shift at the same position) appearing more than once. Is there a way to circumvent setting a RT Prediction algorithm and just use the settings/times from the document grid list?

Thanks a lot in advance!

Regards,
Jens