files with dual polarity

files with dual polarity naymin saw  2022-12-09 07:20

Hi Skyline community,
I am new to Skyline and I found some issues with dual polarities. My .raw files are positive and negative polarities in one file. When I prepare the standards, QC and blank samples in positive mode, it worked well.
However, when running in negative mode, there is no chromatogram found.
Could you please advise how to work with these dual polarities .raw files? Or please check the transition lists I have prepared and let me know if anything is not correct.

Many thanks,
Nay Min

Nick Shulman responded:  2022-12-09 07:25
Can you send us your Skyline document and one or more of your raw files?
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 and your .raw file(s) are less than 50MB you can attach them to this support request.
You can upload larger files here:

I cannot tell from your screenshots what might be going wrong but after we see your files we will probably be able to figure it out.
-- Nick
Brian Pratt responded:  2022-12-09 08:22
From the pictures there's nothing obviously wrong with your transition lists, but it would be helpful to have those files as well.


Brian Pratt
naymin saw responded:  2022-12-12 00:23
Thanks for your responses. I have attached the Skyline doc and sample raw files too.
Nick Shulman responded:  2022-12-12 10:28
I see that at "Settings > Transition Settings > Full Scan" you have MS1 filtering enabled, but do not have MS2 filtering enabled.
That means that Skyline will only be extracting chromatograms for the precursor.

In order for something to be recognized as a precursor by Skyline, the precursor and product m/z's need to be the same number.
In the attached screenshot, the precursor is 164.0717 and the product is 166.0863, so Skyline would only extract chromatogram points for that transition from MS2 spectra, but MS2 chromatogram extraction is disabled by the settings at "Settings > Transition Settings > Full Scan".

Do you know which is the correct m/z for that molecule?
If you want to add precursor transitions to your document, you would usually specify the precursor formula and the product formula in the same way.
In your transition list, the precursor is specified with the formula C9H11NO2, but the product is specified with the m/z 166.0863. If those two things are supposed to be the same, then I would recommend that you remove the column "Product m/z" and instead have columns "Product Formula" and "Product Adduct" and put the same things in those two columns as you have in the columns "Precursor Formula" and "Precursor Adduct".

It's possible that I am misunderstanding what you want your transition list to look like. If you were hoping to get MS2 chromatograms, let me know what you were hoping your transition list would be.
-- Nick
Brian Pratt responded:  2022-12-13 11:45
Looks like you're using the protonated m/z rather than the deprotonated m/z in your negative transition list.You can just get rid of the product m/z and product charge columns and Skyline should do the rest automatically based on the chemical formula. Also, note that you don't have to put the positive and negative lists in separate documents if you don't want to.

- Brian
naymin saw responded:  2022-12-15 00:41
Thanks Nick and Brian. File reading is working now. I am trying to read only MS1 Full Scan. I have changed the Precursor m/z.

Additional question
As Brian suggested, I don't want to prepare the positive and negative lists in separate documents. But will skyline read the transition list for both of them at the same time? How can I prepare the format?

Nay Min
Nick Shulman responded:  2022-12-15 01:04
Yes you can have positive and negative ions in your document at the same time.
Each precursor ion must have the same polarity as its product ion.
Use the "charge" or "adduct" columns in your transition list to tell Skyline the polarity of the ions.