Library versus DDA import for PRM

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Library versus DDA import for PRM abrahampe  2018-02-21 11:30
 
Hello,

I generated DDA data on a QExactive Pro instrument that I then used to build a spectral library from Crux results. I have created a .ssl file as instructed here (https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BiblioSpec%20input%20and%20output%20file%20formats).

I imported the .ssl and created a library that appears to be correct.

Next, I then imported the results (.mzML) files that were used to generate the library. Interestingly, the peak picking (transition settings: MS/MS filtering, acquisition method=targeted, orbitrap, resolution 60k @ 200 m/z) does not seem to be working properly. I would expect the dot product scores to be a value near 1, given that the library was generated from the same data. Oddly, the peak picking retention times are, in many cases, nowhere near the ID time. For whatever reason, Skyline isn't identifying the transitions near the ID time. I have used the parameters outlined in the webinar document and I've looked through the MS/MS manually and the mass tolerances are well within the set parameters. I'm not quite sure what's happening. I've looked at previous posts regarding this topic and so I've made sure to check file names, for example.

Thoughts?

I am using Skyline 64bit version 4.1.0.11796.

--
Paul

 to obtain a refined list of precursors that will be used for a PRM approach.

I am using the Webinar-17 document as a reference.
 
 
Nick Shulman responded:  2018-02-21 14:45
Can you send us your files?
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 and spectral libraries.

You can upload that .zip file here:
https://skyline.ms/files.url

You should also send us a few of your raw files.

I am not sure how good we would expect the library dot product to be. I could imagine that they might be a little different because Skyline is looking at integrated chromatogram peak areas, and the spectral library has the intensities from a single MS2 scan.
 
abrahampe responded:  2018-02-22 07:00
Hello,

I have tried to import the files, but it says that I do not have privileges to upload.

--
Paul
 
Brendan MacLean responded:  2018-03-06 17:58
Hi Paul,
Did you manage to get files to us? We have run into this multiple times now. If you get an error about not having privileges, that means there is already a file by the name you are using uploaded, usually because you already successfully uploaded it. We are working to get a better message for this.

Thanks for helping us understand your issue.

--Brendan
 
abrahampe responded:  2018-03-07 06:04
Hi Brendan,

Thank you for the response. Yes, I was able to get them to upload and you are correct that the issue was that I didn't realize it had already successfully uploaded. My connection was pretty poor that day.

The requested files to troubleshoot my initial issue are listed under "PRM_Settings_CJ473_reduced.sky"

Regards,
Paul
 
Nick Shulman responded:  2018-03-09 15:02
Hi, Paul,

It looks like you are trying to extract MS2 chromatograms from DDA data.
That produces awful-looking chromatorams (with long straight lines) because any particular precursor does not get sampled on any sort of consistent schedule.

With DDA data, you should only use MS1 chromatograms.

You might want to look at the Skyline DDA MS1 tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_ms1_filtering