problems in creating new equation for CE amanzoor  2021-08-23
 

Dear Skyline team,

Thanks a lot for your help. We just started to use skyline for targeted proteomics. Everything is fine till we reach where we have to calculate the linear equation for CE. Although we can see nice peaks with good intensity but the graph is not up to the mark. I have attached the screen shots of our skyline and the graph. Please find it attached. We are using Skyline (64-bit) 20.1.0.155 version on windows 7. Please let me know if you want to have some more informations.

The other thing I would like to ask that there is no internet in our computer. Do you have some system to upgrade our skyline version offline?

Thanks in advance,
Looking forward hearing from you
Best regards
AMK

 
 
Nick Shulman responded:  2021-08-23
I am not sure I understand your question collision energy regression question, but if you send us your Skyline document we might be able to figure it out.

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

If you need to install Skyline on a computer which is not attached to the internet, then you should use the "unplugged" installer:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=install-64-disconnected_21_1

The unplugged installer does not know how to automatically check for updates, so I think you probably need to go into Control Panel > Programs and Features and uninstall the old version of Skyline and then run setup.exe from the new unplugged installer that you have downloaded.

-- Nick
 
amanzoor responded:  2021-08-24
Dear Nick,

Thankyou very much for your reply. I have uploaded the skyline file under the name skyline_AMK and also attached it with this mail. Please find it attached.

Thanks once again

Looking forward hearing from you.

Best regards

Asif Manzoor
 
Nick Shulman responded:  2021-08-24
Thanks for sending those files.

I think everything is working well. I see that you have a replicate called "CE" which was collected data for multiple collision energies.
If you want Skyline to create an optimization database (.optdb) using the results in that replicate to specify the best collision energy to use for each peptide and transition, you can go to:
Settings > Transition Settings
and choose "Add" from the "Optimization Library" dropdown.

In the screenshots that you sent with your original question, you showed the results of adding a new collision energy equation. I am not much of an expert on collision optimization in Skyline. I am not sure whether it is to be expected that your points should be spread all over the graph like that, with no obvious relationship between precursor m/z and collision energy. I imagine that you will get the best results if you use one of the built-in equations

I imagine everything will work best for you if you use the "TSQ Quantiva" collision energy settings which come with Skyline, since that is the type of mass spectrometer that your data was collected on.
-- Nick
 
amanzoor responded:  2021-08-25
Dear Nick,

Thank you for your reply. I am attaching the protocol which we refer to while running the skyline. In page 9 and 10 of the protocol we use to calculate the equation coefficients. In the example from the protocol it comes in positive slope or intercept but in our system it comes in negative slope or intercept. We don't understand this. it is asking to delete some of the points where the peaks are very nice. May be it is due to older version we are using? Is it easy if we can talk on phone or you can connect me who has a good experience with CE.

I will really appreciate your help

Thanks a lot

Best regards

Asif M