Number of scans from raw data ulisse cucchi  2021-07-29 06:46
 

Hello Skyline Team,

I'm still trying to learn the basics of the software, in particular for the targeted analysis of peptides by PRM. I managed to perform all the steps well described by your tutorials, but I have an issue regarding the number of scans that are imported into Skyline from the raw data. To show you the issue I attached you 3 slides in a pdf file.

In the slide 1 you can see the peak from the raw file (two different visualizations of the same peak are showed). As you can see the peak contains over 40 scans of the same precursor (the instrument used was the Orbitrap Exploris 240 from Thermo).

In the second slide you can see how the same peak is imported into Skyline. As you can see, only few scans (less than 10) are used and the resulting peak has a lower resolution, that means also a lower accuracy when the peak is integrated.

In the third and last slide you can see the only parameter that I've found that should affect the number of scans that are imported, but the option "include all matching scans" was already selected. Since they are all very good scans from the raw file, I don't understand why Skyline is considering less than 10 of them. Is there any way to make Skyline import all the scans present in the raw file?

Thanks in advance for any answer and suggestion you may provide,

Best regards,

Ulisse Cucchi from Nerviano Medical Sciences (Italy)
 
 
Nick Shulman responded:  2021-07-29 07:37
Can you send us your Skyline document and raw file?

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 and your raw file are less than 50MB you can attach them to this support request.

Otherwise, you can upload those files here:
https://skyline.ms/files.url

When your isolation scheme is "Targeted", a particular MS2 scan will only contribute to the chromatograms of the precursor in the Skyline document is closest to what was isolated in that scan. Sometimes when your chromatogram does not have as many points as you would expect, the problem is that some other precursor in your Skyline document has a closer m/z to what the mass spectrometer says was isolated. If your intention was that some MS2 spectra were meant to be used by more than one analyte in your document, then you should set the acquisition method to "DIA" and choose something such as "Results Only" for the isolation scheme.

The "include all matching scans" option affects how long the chromatogram extends beyond the peptide's predicted retention time. That setting would not affect the number of scans that you are seeing across that peak in your screenshot.

I am not sure what is going wrong for you, but after I see your .sky.zip file and your .raw file I will probably be able to give you a better answer.
-- Nick
 
ulisse cucchi responded:  2021-07-29 08:23
I uploaded the file generated from Skyline and the raw file, as requested.

The analysis was performed in PRM mode, scanning only one precursor within that time range. No other precursors were fragmented in this analysis (it wasn't a DIA). I see more than 40 scans of the selected precursor in the raw file using Freestyle software, but only few of them are visible in Skyline. Maybe that is the way it is supposed to work.

Ulisse Cucchi
 
Nick Shulman responded:  2021-07-29 08:36
Thank you for sending those files.

You should right-click on your chromatogram and choose "Transform > None".

In your original screenshot, Skyline is showing the interpolated chromatogram. Skyline's peak detection algorithm only works on chromatograms whose points are evenly spaced in retention time. The first step that Skyline does for peak detection is to choose a time interval, and then resample the chromatogram so that all of the chromatogram points are evenly spaced. By default Skyline shows you the interpolated chromatogram, but you can switch it to the raw chromatogram with the "Transform > None" menu item.

For most of the length of that chromatogram, spectra were collected once every second. At the spot where the chromatogram intensity was higher, it appears that the mass spectrometer was able to collect spectra 20 times faster than that.

Skyline chose a time interval for interpolation which was typical of the entire length of the chromatogram, but which was much smaller than the sampling rate in the region that you are interested in.

We tried to implement our interpolation algorithm so that differences like this would not have any impact on the peak areas that get reported to you.

I hope this answer makes sense.
-- Nick
 
ulisse cucchi responded:  2021-07-30 01:26
Thank you Nick. The answer makes a lot of sense.

best regards,

Ulisse