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.

--Brendan

Screenshot should be in this response normally.

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

https://skyline.ms/files.url

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

Thanks for reporting the issue.

--Brendan

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

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

Best,

Brian Pratt

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!

Brian

Works like a charm!

Thx a lot.