Retention time shift

Retention time shift yulun  2017-04-28
Hi all,
I run some standards (metabolite) in different runs and would like to analyze by Skyline. Please see the attachment. The most right panel is the correct peak identified (~13.5 min) by the Skyline. However, the most left (1.5 min) and the middle (22.8 min) ones return the wrong retention time. I know the Skyline did the good job to pick the highest peak. My question is: Is there any possible way from setting to get rid of these 2 peaks identification since it is out of rt windows (0.2 min for rt window)?
Thank you
Nick Shulman responded:  2017-04-29
I do not understand your question. Usually a question about incorrect peak identification would have a picture of a single chromatogram with several peaks. You have a picture of three chromatograms and each of those chromatograms has only one peak.

Maybe it would help if we could see your Skyline document.
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, you can attach it to this support request.

If you want to learn more about the criteria that Skyline uses for picking peaks, you can take a look at the Advanced Peak Picking Models tutorial:

Also it might be helpful for you to specify a value in the "Explicit Retention Time" column in the Document Grid for your analyte.
yulun responded:  2017-04-29
Hi Nick,
This is a standard sample and we run three different methods which show in three chromatograph. Only the most right method return the reasonable RT (~13min). The other two methods detect the wrong RT. We have use 'Ecplicit RT' in the document Grid.

My question is if there is any way to force Skyline not report the peak because of the its RT is out of reasonable time frame?

Thank you. I will upload the zip file next Monday.
Nick Shulman responded:  2017-04-29
It might help to do something with the "Retention Time Filtering" settings on:
Settings > Transition Settings > Full Scan
If these are extracted ion chromatograms, then you can tell Skyline to limit the time range over which the chromatogram is extracted.

Is there a reason that you cannot just manually adjust the peaks to where they should be, or remove the peak ("Remove Peak" on the right-click menu on the chromatogram)? Do you have too many samples for it to be possible to manually adjust or remove peaks?
yulun responded:  2017-04-29
I will try "Retention time filtering". Yes, we have many standards and several methods. So we just wonder if there is more efficient way to do it.

BTW, is there any video about the manually peak selecting or zoom-in the chromatograph? Since we only have had 2 months experience on using skyline.

Thanks Nick
yulun responded:  2017-05-01
I tried RT filtering but it does not work out.
The .zip was attached.
Brendan MacLean responded:  2017-06-08
Hi Yulun,
Sorry for the long pause in communication. I guess we got distracted by ASMS.

When I look at the document you have provided, your RT filtering appears based on MS/MS IDs, which we don't even support for anything but peptides.

You would need to make the filtering window based on RT "prediction", using something like "Use scans within [ 1 ] minutes of predicted RT", instead of "Use scans within [ 1 ] minutes of MS/MS IDs" as you have.

It looks like you have already set up your Explicit Retention Time values, which are what Skyline will use as the "predicted RT" so that is good, but you have also entered as Explicit Retention Time Window values 0.5 for all. This seems to have backward the concept of a "window" and "within". The term "within" is indicating +/-, while window is the entire range:

1 minute window = within 0.5 minutes (i.e. +/- 0.5 minutes)
2 minutes window = within 1 minute (i.e. +/- 1 minute)

While what you have:

0.5 minutes window != within 1 minute (i.e. +/- 1 minute)

In this case, I am not even 100% sure which Skyline will use. I would expect it to favor the Explicit Retention Time Window over everything else and use only the "within" value for molecules where you do not supply the Explicit value. But, you could ensure that by clearing the Explicit values altogether, since they are all the same. Then if you really wanted a 0.5 minute window, you would use within 0.25 minutes.

Hope this is not too confusing. It looks like at least you are only 1 step away from seeing your chromatograms filtered. All you need to do is change to "within minutes of predicted RT". Then you just need to work out exactly how much time you want in your chromatograms.

Yasin responded:  2018-08-02

I hope that someone is still following this old post (otherwise I iwill create a new one tomorrow) but I have a very similar problem.

I have set explicit Retention times in my transition lists (no value for the explicit retention time window) and checked the retention time filtering to within 0.2 minutes of predicted RT. However when I try to load result files for processing into that skyline method I get an error message saying that no prediction algorithm has been specified...

Would be happy if somebody could help with that problem. Thanks a lot in advance!

Nick Shulman responded:  2018-08-02
If you have specified the Explicit Retention Time for any peptides or molecules, and you want Skyline to use that number to limit the length of the extracted chromatograms, then you should choose the "Use only scans within X minutes of MS/MS IDs" setting on "Settings > Transition Settings > Full Scan > Retention Time Filtering".

The "within X minutes of predicted RT" setting does not look at the Explicit Retention Time.
Nick Shulman responded:  2018-08-02
Actually, I believe what I just said was not correct. Let me get back to you in a few days about how Explicit Retention Time is supposed to work with Full Scan retention time filtering.
Yasin responded:  2018-08-03
Ok, thank you a lot for your effort!
bryna fitzgerald responded:  2020-08-19
I am also having a similar problem when I am trying to target small molecules based on MS1 scan. I have specified explicit retention times in my transition list, but when I try to use "use only scans within X minutes of predicted RT", I get the same error as Yasin. Is there a way to have it look at the explicit retention times? Or is there another way to provide a retention time window based on explicit retention times?

When I use the "include all matching scans", it mostly picks the right peak, but there are times when it is way off. I have a lot of small molecules and a lot of samples that I am targeting, so it is difficult to go through and manually evaluate every one.
Brian Pratt responded:  2020-08-19
Hi Bryna,

What's the version number of the Skyline you're using?


Brian Pratt
bryna fitzgerald responded:  2020-08-19
Currently I am using Skyline-daily, but I just updated and was using a slightly older version from a couple months ago when I encountered this issue.
Brian Pratt responded:  2020-08-19
I'm unclear on whether or not you are experiencing this issue with the current version of Skyline.

We did make a change in the Explicit RT behavior not long ago: formerly Skyline preferred any peak found within the explicit RT window, but would still seek out a peak elsewhere if none was found within the window. Now it simply doesn't record a peak if the explicit RT window doesn't contain one. Hopefully that resolves your issue.


bryna fitzgerald responded:  2020-08-19
Thank you!! I feel silly, I didn't realize that there was an option for an explicit retention time window column in the transition list, but I see that now. Sorry to bother you and thanks for your help!