Yes, Skyline works great with negatively charged ions.
In a transition list, you can indicate that an ion is negatively charged by specifying a negative number in the "Charge" column. The m/z values should always be positive numbers regardless of the charge on the ion.
-- Nick

Hi Nick,

Thanks for your prompt reply.

As you suggested, I tried. But I could not see m/z values corresponds to negative ions.

Kindly refer attached sheet (slide number 4).

Regards
Dilip

Oops. I missed the fact that you were talking about peptides.
With peptides, Skyline only allows you to use positive charges.
If you want to use peptides with negative charges, you would need to tell Skyline the chemical formula of those peptides.

If you go to "View > User Interface > Mixed", then Skyline will allow you to have both peptides and small molecules in your document at the same time.
After you have switched to mixed mode, you can go to the Document Grid and look at the report called "Small Molecule Transition List".
The chemical formulas in that transition list might be a good starting point for building the transition list that you will eventually import into Skyline.

I do not know much about how peptides behave when they are negatively charged, so I do not know what tweaks you will need to make to those chemical formulas for them to be negatively charged.
-- Nick

Thanks, Nick!

I will check in mixed mode and let you know if face any difficulty.

Regards
Dilip

Just a note to say that we only deal with protonation for peptides because other ionizations like sodiation or deprotonation can result in fragmentation behavior that Skyline doesn't know how to model. So, as Nick says, you'll have to come up with an explicit set of formulas (or at least m/z values) to describe the precursor and fragment transitions.

-- Brian