IS it possible to do PRM data analysis using skyline? ekramh1982  2019-09-26
 

The format (column headings) for the inclusion list of PRM in Q Exactive plus is different from the example provided in "HiResMetabolomics.zip" file. And creating a file similar to the values gets this problem: when I import the raw files it says "no usable data". How do I quantify the PRM files from Q Exactive Plus using skyline? The message is as follows:

At 11:52 PM:
Failed importing results file 'D:\Ekram\2019\Sept_20\Swab_Recovery_PRM\Pos_SwabRecovery_PRM\Pos_PRM_P40_Tang_100uM_stock_A10.raw'.
The sample Pos_PRM_P40_Tang_100uM_stock_A10 contains no usable data.

But I can open that file in quail browser: https://imgur.com/RSjtZGg

How do I solve this? Thank you.

 
 
Brian Pratt responded:  2019-09-27

This will be easiest to figure out if we have your files:
The transition list you fed into Skyline
The .raw file
The Skyline document (use the Skyline File > Share > Complete menu item to create a .sky.zip file)

Upload those to https://skyline.ms/files.url and we'll have a look. It's probably something simple. (note: the site will be down over the weekend for maintenance)

Thanks for using the Skyline support board!

Brian Pratt

 
ekramh1982 responded:  2019-09-27

I uploaded the files with description as "ekramh1982_PRM_Issue"

 
Brian Pratt responded:  2019-09-27

It looks like the .raw file contains only MS2 scans. As Skyline has only been given precursor targets, it can't do anything with that file. I'll see if we can't make that error message a bit more informative in this case.

Can I see the original inclusion list?

Cheers
Brian

 
ekramh1982 responded:  2019-09-27

Here is the original inclusion that I ran on the QE+

 
Mike MacCoss responded:  2019-09-27

The issue is that you need to tell Skyline which product ions to create extracted ion chromatograms of.
-Mike

 
Brian Pratt responded:  2019-09-27

Skyline is designed for targeted mass spec, so you'll have to come up with some fragment targets to proceed. Do you have access to any spectral libraries such as NIST?

 
ekramh1982 responded:  2019-09-27

When I run PRM the inclusion list only asks precursor, not transition list. Hence there is no MS1 in the scan. How to process these type of files then? Thank you.

 
ekramh1982 responded:  2019-09-27

No, I do not have any access to NIST database directly. I do have access to GNPS (gnps.ucsd.edu) server and the database there. Will that help?

 
Mike MacCoss responded:  2019-09-27

You're correct that to collect an MS/MS spectrum (what PRM is doing), you only need to select a m/z for your precursor selection and the amount of energy that you want to supply. However, to extract a precursor > product ion chromatogram, you need to give it a precursor and product ion pair to extract. For peptides we can usually predict likely product ions but this is harder for non-peptide molecular species you will want to have data from either a standard or some prior measurement where you are confident the spectrum reflects the target molecule of interest.

I will also point out that it is usually best to start your method development in Skyline itself. It is designed to help with the method development and analysis. I would suggest looking through the tutorial on PRM https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_targeted_msms. Even though it is for peptides many of the aspects are the same.

You can also look at the small molecule targets tutorial (https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_small_molecule). The method is for a triple quad but the process is the same except in this case you need to tell the QQQ instrument what m/z values to measure for the product ions first. In PRM you collect the entire spectrum so you can collect all m/z values and extract the data after the fact.

There are some great webinars too:
https://skyline.ms/project/home/software/Skyline/events/2017 Webinars/Webinar 16/begin.view?
https://skyline.ms/project/home/software/Skyline/events/2018 Webinars/Webinar 17/begin.view?

Yes, you should be able to use gnps data to help pick product ions. However, just remember that the product ions will be dependent on the instrument platform and collision energy used.

Best of luck in your experiments. Hope this helps,
Mike