1. If you want Skyline to specify two different Explicit Collision Energy values for the same molecule in your Skyline document, you should add the molecule twice (on "Edit > Insert > Transition List"). Furthermore, these two molecules should be given different "Precursor Name" values.

2. For MS2 chromatograms, Skyline only extracts the chromatogram for the monoisotopic mass of the fragment ion. If you would like Skyline to extract one of the other other isotope peaks, then, you should specify the "Product m/z" that you want. You should also make sure that the Product Formula is blank.
-- Nick

A little clarification on point 1: Nick is describing a way to specify different CE values when Skyline is writing a transition list, but I think what you are asking about is comparing two different mass spec runs that used different tuning parameters. So you can disregard that part of his answer. (It's also worth knowing that Skyline has automated CE optimization to help you find the best CE for each transition.)

Nick's second point is worth noting if you are interested in unfragmented precursors in MS2 scans. Chromatogram extraction of M+1, M+2, etc in MS1 data is automatic.

Hi Nick and Brian,

Thanks for your response!
For the 1st question, we want to optimize the MS conditions for a target peptide simultaneously (in 1 run but different transitions, as shown in the attached figure). However, Skyline seems unable to show the results of different MS settings with the same precursor and product ion. So we have to check every transition in the raw file and record the intensity, then we can plot the data to get the most intensive product ion and optimal CE and tube lens.
For the 2nd question, some times we want to look at different isotopic SRM transitions, such as M-M, M-M+1, M+1-M, M+1-M+1. For example: a precursor with a m/z 593 and its charge state is 2, and the most intensive product ion is 846 with charge state 1. We want to compare 593- 846, 593-847, 593.5-846, 593.5-847.
Can skyline accomplish the above mentions problems?
Thanks!

Best,
Chao

Hi Chao,

>> optimize the MS conditions for a target peptide simultaneously (in 1 run but different transitions, as shown in the attached figure).

For SRM, Skyline doesn't do this in a single run, but it does provide excellent tools for comparing runs.

>> some times we want to look at different isotopic SRM transitions, such as M-M, M-M+1, M+1-M, M+1-M+1

You could certainly create a, SRM transition list like this and import it into Skyline, but creating it automatically isn't something Skyline does.

I hope this helps.

Best,

Brian