• Sign In
  1. Requests

Get chromatograms for few peptides

support

View Request

view list print
Get chromatograms for few peptides shubham1637  2023-09-30 17:28
 

Hi,
I have DIA files from small-amount samples. I did not spike-in iRT standards. Is there a way to get/see chromatograms for few candidate peptides? I am trying to load file in Skyline, however, since there are no iRT peptides, it is failing. I also tried CiRT but that also failed.

Thanks!

At 8:27 PM:
Failed importing results file 'F:\Users\rona\DIA-QE\DIA-QE\DIA\collinsb_X1803_171-A.mzML'.
The calculator test1-assay requires all of its standard peptides in order to determine a regression.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'F:\Users\rona\DIA-QE\DIA-QE\DIA\collinsb_X1803_171-A.mzML'.
The calculator test1-assay requires all of its standard peptides in order to determine a regression. ---> pwiz.Skyline.Model.Irt.IncompleteStandardException: The calculator test1-assay requires all of its standard peptides in order to determine a regression.
at pwiz.Skyline.Model.Irt.RCalcIrt.ChooseRegressionPeptides(IEnumerable`1 peptides, Int32& minCount) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Irt\RCalcIrt.cs:line 152
at pwiz.Skyline.Model.Results.ChromCacheBuilder.RetentionTimePredictor.CreateConversion() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 838
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 404
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252
--- End of inner exception stack trace ---
 
 
Nick Shulman responded:  2023-09-30 18:15
The reason that Skyline is insisting that you have iRT standards is that your document has specified that it is using an iRT predictor.

If you don't want to use iRT with this document you should go to the "Prediction" tab at:
Settings > Peptide Settings
and change the retention time predictor to "None".

After you do that you will be able to tell Skyline to extract chromatograms using the "File > Import > Results" menu item.
--Nick
 
shubham1637 responded:  2023-09-30 19:38
Thanks Nick. It worked.
I was also wondering if there is any intensity cutoff? as I am working with small-amount sample, I see many traces empty.
 
Nick Shulman responded:  2023-09-30 20:14
Skyline does not do any sort of intensity cutoff, but mass spectrometers sometimes do (it's a way to reduce the size of the raw file).

You might find some useful information about how Skyline calculates background here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tip_peak_calc

If you click on a point along the chromatogram, Skyline will display the spectrum that contributed to that point. That might give you some insight into why the value is what it is.

By the way, I noticed from the call stack in your original post that you are running Skyline version 20.2.

The current version of Skyline is 23.1.

Some people use old versions of Skyline if they are trying to reproduce results in old experiments but let us know if anything is preventing you from being able to upgrade to the latest version.
--Nick
 
shubham1637 responded:  2023-10-02 14:16
Thanks for your help. I have two more questions:
1. I am looking at chromotograms using Assay library. However, I only see fragment-ion traces. Is it possible to enable overlaying MS1 traces?
2. When I click on a point on chromatogra, I see spectrum in the pop-up window. When I hit -> or <-, it goes to the second next point. I was wondering is Skyline generating intermediate point? or is it intentionally skipping the point?

Shubham
 
Nick Shulman responded:  2023-10-02 14:35
I am not sure that I understand your first question.
Can you send us your Skyline document?
In Skyline you can use the menu item:
File > Share
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, otherwise you can upload it here:
https://skyline.ms/files.url

For your second question, you should right-click on your chromatogram and choose "Transform > None".

Skyline's peak detection algorithm involves first resampling the chromatogram points so that the retention times of the points are evenly spaced in the time dimension. With this interpolated chromatogram, the points displayed on the graph do not actually correspond to the spectra that were actually collected. If you change the "Transform" to "None", then every vertex on the chromatogram graph will correspond to a real spectrum.

When you are looking at the spectrum viewer of spectra from the chromatogram, the list of available spectra is dependent on the Transform setting on the chromatogram graph. When looking at an interpolated chromatogram graph, the spectrum viewer will occasionally skip over spectra when you use the forward and back buttons.
-- Nick