Fail to import Thermo raw PRM data

support
Fail to import Thermo raw PRM data haidi yin  2019-03-13
 

Dear Skyline team,

I used to import Thermo raw PRM data successfully. I use a portable hard disk to store my raw data. I can still open them with Xcalibur.

But these days I fail to import those old raw data to skyline daily. It did show chromatogram during importing. But after importing, no error sign or chromatogram is shown, as if the raw data was not imported. Attached is the skyline interface after importing one raw PRM data. Do you have any idea what went wrong?

Thank you very much,
Best regards,
Haidi

 
 
Nick Shulman responded:  2019-03-13
That looks like what would happen if there were no MS2 scans that match your precursor.
That is, you have no MS2 scans whose precursor is either 1007.4005 or 1056.0865.

What do you have selected on
Settings > Transition Settings > Full Scan > MS/MS filtering Acquisition Method
?

If that's "Targeted", then the setting that determines how close the MS2 scan precursor m/z has to be to your peptide's m/z is determined by the "Method match tolerance m/z" setting at:
Settings > Transition Settings > Instrument

If you send us your .raw file and Skyline document we can figure out what is going wrong.
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that .zip file and your .raw file are each less than 50MB, you can attach them to this support request.

Otherwise, you can upload them here:
https://skyline.ms/files.url
-- Nick
 
haidi yin responded:  2019-03-13
Dear Nick,

Thank you very much for your answer. But I am very sure there are MS2 scans of 1007.4005 or 1056.0865. I set PRM on them. And I succeeded to import them several days ago.

The zip file is very small, please find in the attachment.

Best regards,
Haidi
 
Nick Shulman responded:  2019-03-13
Haidi,

Thank you for attaching that .sky.zip file. I think I need to see your .raw file in order to actually know what is going wrong. It looks like Skyline did not find any matching MS2 spectra in your .raw file, but I cannot know for sure without seeing the .raw file.

Can you send me that file "C:\Data\20190222_A1AT PRM\20190222_stdA1AT_PRM_newgradient.raw"?
You can upload it here:
https://skyline.ms/files.url
-- Nick
 
haidi yin responded:  2019-03-13

Dear Nick,

Thank you very much for the prompt response!

Could you please download the raw file from one drive: https://polyuit-my.sharepoint.com/:i:/g/personal/hdyin_polyu_edu_hk/EZnNYGj9yDBClN4p88TrL-IBbxdnmgwPpCvZlRuS52ugXQ?e=yrAdMM

Best regards,
Haidi

 
Nick Shulman responded:  2019-03-13
The precursors in your Skyline document are:
1007.400506
1056.086476

The closest MS2 spectra in your .raw file are:
1007.7433
1056.4305

If you want Skyline to use those MS2 spectra, then you should go to:
Settings > Transition Settings > Instrument
and change "Method Match Tolerance" to a higher number, such as "0.4". You currently have that set to "0.05".

It looks like whatever you used to generate your PRM method disagrees with Skyline about the neutral mass of your peptides are. The difference between what Skyline is expecting and finding is about .33 on a charge 3 peptide, which is close to a 1 Dalton difference in the neutral mass.

If Skyline is getting the wrong answer, then it might be because the chemical formula of the modifications is missing a hydrogen.
Your two modifications are:
name="54200" formula="C84H136O61N6" (mono mass 2204.77)
name="54201" formula="C90H146O65N6" (mono mass 2350.83)

If those formulae are wrong, you can fix them by going to:
Settings > Peptide Settings > Modifications > Structural Modifications Edit List..
-- Nick
 
haidi yin responded:  2019-03-13
Dear Nick,

Thank you very much. Oh, it is the isotope problem. I may have used a bigger Method Match Tolerance previously.
Now it is settled! Thank you!

Haidi