Define wich trace to proces

Define wich trace to proces stsyp  2019-03-05

Hey Skyline

I was trying to use Skyline to analyze SmallMolecules. The .raw file I have consist of 3 traces: neg mode ionization, pos mode ionistation and a PDA trace.
I have the impression that Skyline is only analyzing the 1st trace (pos mode). Is there a possibility to define which trace skyline needs to process? That for example trace 2 is used to analyze the compounds in MoleculeListName "X" and trace one is used to analyze the features in list "Y"
I've added a ppt to make clear what I try to explain.

I hope this is already possible but I just don't find how to apply it.

Steven Vds

Brendan MacLean responded:  2019-03-05

Can you include a screenshot with the molecules expanded so that we can see how you have defined the ions?

It is possible to define negative mode ions in Skyline by using negative charge states (e.g. -1) or adducts like [M-H]. So, it would seem that you do not have the ions correctly defined for Skyline to be able to match what it finds in your raw data file.


stsyp responded:  2019-03-05

Screenshot should be in this response normally.

Brendan MacLean responded:  2019-03-05

Interesting. Those look defined correctly to me. I think we will need your Skyline document (use File > Share - Complete to save a file) and a raw data file, which you can upload to:

For us to have a closer look at why this is not working as expected.

Thanks for reporting the issue.


stsyp responded:  2019-03-05

Hey Brendan
The two zip files are uploaded to your servers. I hope you can reproduce the problem, and more importantly, find a possible solution ;)
Steven Vds

Brian Pratt responded:  2019-03-05

Thanks for the files. Your document seems proper, so there's apparently something unusual about the raw data that, we'll look into it.


Brian Pratt

Brian Pratt responded:  2019-03-06

OK, here's what I've found:

Those .raw files are unusual in that they contain some data that's not MS1 or MSMS. That's only a problem if you want to centroid the data, we have alerted Waters to the problem.

That's OK, though - you weren't using centroiding anyway. The issue with your Skyline document is that you're trying to extract DIA data where there is none. Skyline takes this DIA setting as a hint that the .raw file is msE data, so the function that's actually the negative scans is treated as the high energy msE data channel.

So, in your Full Scan settings, just set MS/MS filtering's Acquisition mode to None (you had it as DIA), and in the Filter tab set the Small Molecules Ion Types to "f,p" so that precursors are active, then reimport.

Thanks for using the Skyline support board!


stsyp responded:  2019-03-08

Works like a charm!

Thx a lot.