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"No Valid Precursor m/z column found…" sa660  2018-01-24 08:52
 
Hi,

My name is Shimon and I am a 3rd year BSC student with Professor Don Jones at the University of Leicester. I'm trying to import 3 sets of transitions from Mass Lynx Version 4.1 into Skyline. 2 of the transitions have successfully been imported but the third has not. The one that isn't being imported is highlighted in the Excel screenshot. When I try to import I get the error: "No Valid Precursor m/z column found…" shown in the 2nd screenshot. These transitions are from a Waters Xevo MS and both precursor and product ion mass are set to monisotropic. The peptides were produced using a Tryptic Digest

I have restored both the ion and method match tolerance m/z back to the default 0.055 but that doesn't seem to have made any difference, other parameters that I am using that I think might make a difference are shown below:
-    Enzyme = Trypsin
-    No of missed cleavages = 2
-    Structural modifications = Carbamidomethyl (C)
-    Max Variable mods = 3
-    Max neutral loses = 1
-    Isotope label type = Heavy


I hope you can help.

Thanks,

Shimon
 
 
Brendan MacLean responded:  2018-01-24 16:23
Hi Shimon,
Can you please supply the Skyline document (using File > Share - Complete to create a .sky.zip file) and the text transition list? They should be pretty small. So, you can just attach them to this thread.

If I type out the first row of the transition set I think you are talking about, it pastes into Skyline just fine for me.

I would prefer not to have to type out everything, and it maybe be something in your settings that I do not have. Saving a file that you are pasting into will ensure that I have the same settings as you.

Thanks for posting to the Skyline support board.

--Brendan
 
sa660 responded:  2018-01-25 01:00
Good morning Brendan,

Here are the files. When sharing the Skyline document, it offered me to share formats so I saved both and sent them here.

Thanks,

Shimon
 
Brendan MacLean responded:  2018-01-25 15:54
So, the first step in understanding a failure like this is often to try just to create the precursor of interest in Skyline by simply adding the peptide sequence. When I paste the problem sequence into the document you sent, I get:

LGADMEDVRGR (missed 1)
    609.7984++

Which leaves me wondering where the precursor m/z 610.17 comes from. It appears to be about .37 m/z too heavy.

This is why Skyline is complaining. Because it can't find any value that matches its expected precursor m/z for any charge state.

Can you explain what you are expecting? Or is this just a typo?

--Brendan
 
sa660 responded:  2018-01-26 02:48
Hi Brendan,

You're correct. My value is based on the average peptide mass rather than the monoisotropic (A silly error)
I understand why Skyline won't accept the value now. Is there a way around this in Skyline, a way to still select for the 610.17 precursor?
Or if i change my selection in skyline to the correct value of 609.7984++, would I still get the desired peaks?

Thanks,

Shimon
 
Brendan MacLean responded:  2018-01-26 06:38
Hi Shimon,
There is a setting in Skyline under Settings > Transition Settings - Prediction that will allow you to tell Skyline that your precursor m/z values are based on average masses, but you need all of your targets to be either average or monoisotopic, you can't have a mix. If you did create a mix, then you just have to chalk this up as a loss and use Skyline next time to generate your method for the mass spectrometer. That way you will be sure your m/z values match when you try to import the resulting data.

Thanks for helping investigate the issue.

--Brendan
 
sa660 responded:  2018-01-26 09:11
Alright,

Thank you Brendan