• Sign In
  1. Requests

Analyze Agilent SIM data with Skyline?

support

View Request

view list print
Analyze Agilent SIM data with Skyline? wangqingok  2022-02-02 14:41
 

Dear Skyline team,

We wish to analyze some SIM data collected from Agilent 6495C QQQ instrument. The procedure of importing such data for analysis or ion picking is not very familiar to me. I could not find a tutorial showing this either. Can you help? Thank you so much for your great help!
 
 
Nick Shulman responded:  2022-02-02 14:51
Are you analyzing peptides or are you looking at other small molecules which are not peptides?

For analyzing peptides on triple quad instruments like that, we recommend that you start with either the Targeted Method Editing tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_method_edit
or the Targeted Method Refinement tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_method_refine

If you were doing small molecules which are not peptides, then the small molecule targets tutorial is the one to start with:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_small_molecule

The basic way to use Skyline is:
1. Tell Skyline what molecules you are interested in. For triple quad data, if you are analyzing peptides, that might mean using the "Edit > Insert > Peptides" menu item. If you are analyzing small molecules, you might need to use the menu item "File > Import > Transition List".
2. After you have told Skyline what molecules you are interested in, you use the "File > Import > Results" menu item to read the chromatograms from your mass spec data files (which for Agilent are folders whose name ends in ".d").

Let us know if you're getting stuck somewhere.
-- Nick
 
Nick Shulman responded:  2022-02-02 14:59
I just noticed that you said "SIM data", and not "SRM data".
It is possible that the tutorials that I referred you to are not appropriate for your data.

Can you send us an example of your data files?
You can zip up your .d folder. If that is less than 50 MB you can attach it to this support request. You can upload larger files here:
https://skyline.ms/files.url
-- Nick
 
Nick Shulman responded:  2022-02-03 03:22

Thanks for sending your Agilent .d files.
It sounds like you know a couple of the transitions that you want to monitor, and you are hoping to use Skyline to discover some more transitions.
Skyline is a great tool for extracting chromatograms. Skyline is usually not considered a very good tool for looking at spectra.
The program which we have which is better for looking at spectra is called "SeeMS.exe". That program gets installed when you install ProteoWizard. You can install ProteoWizard from here:
https://proteowizard.sourceforge.io/download.html

You can use Skyline to extract chromatograms from your data.
You mentioned in email that your precursor has m/z 158.1, and your product ions have m/z 141.00 and 95.00.
The way to get that information is copy the following text to the clipboard:

PrecursorMz PrecursorCharge ProductMz ProductCharge
158.1 1 141 1
158.1 1 95 1

In Skyline, you can do:
Edit > Insert > Transition List
and paste in the text that you have on the clipboard.

You also need to tell Skyline that you want to extract chromatograms from MS2. To do that, you go to:
Settings > Transition Settings > Full Scan
and about halfway down
set "Acquisition Method" to "PRM"
and set "Product Mass Analyzer" to "QIT"
and at the very bottom choose "Include all matching scans".

After you have done that, you can save your document and then tell Skyline to extract chromatograms by going to:
File > Import > Results

-- Nick