resolution of MS1 precursors and number of data points

support
resolution of MS1 precursors and number of data points rameshkr  2025-02-21 05:22
 

Hello team

I have acquired MS1 and PRM data on QExactive Plus for small peptides.

  1. Is there a way to extract MS1 peak resolution values using skyline?

  2. Also, what is the right way to get the number of data points across a peak? I exported the peptide transition results but I am getting very different values for same precursor between technical replicates- analysed with same transient time, peak width etc.

Thank you
Ramesh

 
 
Nick Shulman responded:  2025-02-21 09:24
Skyline does not read the mass resolution from the raw files.
If you look at your raw files using software from Thermo (e.g. QualBrowser) I believe you will be able to see what the mass resolution of each spectrum was.
Skyline and the ProteoWizard family of programs (MSConvert, SeeMS.exe, etc) do not read those mass resolution values from the raw file.

If you create a custom report in Skyline, there is a column that you can include on your report "Points Across Peak". The value is calculated for each transition peak. In an SRM experiment, each transition might have a different number of points across the peak, but in a full scan experiment, typically, all MS1 transitions (i.e. precursor isotopes) will have one number of points across the peak for a given replicate, and all of the fragment transitions will have a different number of points across the peak.
You can learn more about custom reports in Skyline here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports

If you have a Transition selected in the Targets tree, you can tell Skyline to show you the retention times where chromatogram points were extracted in between the integration boundaries of the chosen peak. To show those times, right-click on the chromatogram graph and make sure there is a checkmark next to the "Retention Times > Show Raw Times" menu item.
Transitions are the nodes in the Targets tree that have no children. If you have something at a higher level in the tree selected, such as a Precursor or a Peptide then you will not see the retention times of the points across the peak.

If you look at the chromatogram graphs for your different technical replicates it will probably be straightforward to figure out why you are seeing different numbers.

If you would like you can send us your Skyline document and we can take a look.
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.
Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url
-- Nick