transition detection issues

support
transition detection issues clemence balty  2020-12-17
 

Hi,

I am trying to quantify peptides in a biological sample using the light / heavy ratio (using the Sure quant method by thermo). I have designed heavy peptides corresponding to the peptides that I want to quantify in the biological sample. First, I created a spectral library with heavy peptides. Then I added these heavy peptides to all samples (potentially containing the light version of each heavy peptide). When I opened the raw file in Skyline, I detected heavy peptides and their transitions except for two heavy peptides (SGYSSPGSPGTPGSR and TPSLPTPPTR). These peptides are present when I manually check the raw file. I don't understand why skyline can't detect the transitions of these two peptides.

Can you help me or give me advice to solve these problems?
Is the library required to perform quantification?

I can share my raw files and the library I created with you.

Thank you,
Clemence

 
 
Nick Shulman responded:  2020-12-17
Clemence,

Can you send us your Skyline document?

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

It might also be helpful if you could send us one of your .raw files.

By the way, there is a new feature in Skyline 20.2: the "Triggered Acquisition" checkbox on the "Transition Settings > Instrument" tab. This checkbox is intended to work around some peak picking and background subtraction problems that earlier versions of Skyline had with SureQuant methods.

This new feature will probably not help with the problems you are describing, but you can read about this feature here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=TriggeredAcquisition
-- Nick
 
clemence balty responded:  2020-12-18
Hi Nick,

Thank you for your answer. I uploaded the files on the 'files sharing folder' under the name of 'NEURO622_3.raw' and 'SQ_Method_Development_20201120.sky.zip'.

Clémence
 
Nick Shulman responded:  2020-12-18
Clémence,

The problem is that your MS2 spectra have a "scan description" set on them which is not quite in the right format.

In a SureQuant method, the precursor isolation target of an MS2 spectrum never exactly matches the theoretical mass of the peptide because what gets written into the .raw file is what the mass spectrometer observed in the MS1 spectrum which might differ from the theoretical mass by a fraction of a Dalton.
The Thermo SureQuant team observed that if you had a Skyline document with a lot of peptides in it, some of those peptides would have precursor m/z's that were close enough to each other that Skyline would include the wrong MS2 scans with the wrong chromatograms. This could result in chromatograms that had long gaps without any points, or chromatograms that with intensities that jumped around a lot as they oscillated between the scans with small fill time that were meant to measure internal standards, and scans with longer fill time which were supposed to pick up the endogenous peptide.

The solution that Skyline and Thermo came up with was that you would be able to specify in your method that the "scan description" could be set on your MS2 spectra, and then, if you have "Triggered Acquisition" specified on your Transition Instrument Settings, then Skyline would pay attention to the "scan description" values on the MS2 spectra. If the MS2 spectrum had a scan description, and the scan description started with "SQ_", then Skyline would skip over the MS2 spectrum if the scan description was not exactly what it needed to be for the precursor.

In your raw file, the scan descriptions are not quite in the right format. Most of the scan descriptions start with "SQ-", which does not cause a problem, since Skyline is willing to use those spectra for any precursor's chromatogram, since it does not start with "SQ_".

However, for the precursors where you are not getting any chromatograms, the scan descriptions do start with "SQ_", but the rest of them is not quite right. The scan description is "SQ_IS R+10_2" and it really needs to be "SQ_IS_R+10_2" (there is a space where there should be an underscore).

There are a couple of things that you can do to fix this problem:
1. Change your instrument method so that it does not add the "scan description" to any of your spectra and collect your data again.
or:
2. Use ProteoWizard msconvert to convert your raw file to mzML, and then use a text editor to replace "SQ_IS R+10_2" with "SQ_IS_R+10_2".

A couple of suggestions when using a text editor to edit mzML files:
1. mzML files are too large for most ordinary text editors. I use an editor called "emeditor" which can handle files these big text files.
2. If you are going to be editing an mzML file, you should uncheck the box in MSConvert that says "Write Index".

I hope this makes sense and helps.

Let me know if you think that any of the method files, documentation or other things that you downloaded from Thermo might have had a mistake in them. I can let the Thermo team know if they need to fix anything.
-- Nick