When you say "identifying peaks", in which dimension are you asking about? m/z or retention time?

Skyline uses a targeted approach, which means that in the m/z dimension it doesn't need to do any peak identification at all. Skyline just extracts chromatograms around the calculate m/z. If your instrument is well calibrated and high resolution this should work well for very large molecules, until the peaks can no longer be resolved in the m/z dimension. Even then, I have seen tools that do this resolution and create centroided spectra. Skyline also supports extracting centroided spectra based on PPM mass accuracy.

If you mean in the retention time dimension, then I am sure Skyline's peak detection algorithms could be used for this data. But, I suspect from the way the question was phrased, you really mean m/z. Because Skyline uses targeted extraction, it is immune to issues of peak detection in the m/z dimension.

Thanks for posting your question on the Skyline support board. I hope Skyline will prove useful to you in your research.

--Brendan

Thanks for your quick response.

Yes, I am using targeted method (SIM, selected ion monitoring) and it should be m/z dimension. I am also using Xcalibur to process these data. In Xcalibur, I need to choose the peak algorithm method (i.e. Genesis, ICIS, Avalon) to identify and integrate the peaks. And I just realized that peaks are a little bit different with different algorithm methods. So I am wondering what algorithm is used in Skyline for the peak identification and integration.

Because some of my samples are in low concentration, I want to calculate the signal to noise ratio to evaluate if the peak is okay or not. Since different peak algorithms would generate different chromatograms, I am wondering if Skyline has a way to calculate S/N.

Thanks,
Fuyan

The algorithm is to accept spectrum intensities falling within a +/- range around the targeted m/z. The range is either calculated based on the give resolving power for profile data or a PPM value for centroided data. The accepted intensities are summed and the mass error is calculated as their weighted mean delta-mass from the targeted m/z.

Does that help?

Yes. Thanks for the details.

Fuyan

I have another question. Each compound has three adducts added. Is there any way to combine these three peaks into one?

Thanks,
Fuyan

There is a way to combine them into a single neutral molecule that will force integration to use the same retention time boundaries. The peak areas for these will each have their own background subtraction. You should be able to sum those peak areas pretty easily, though.

You can look at the Skyline tutorial Existing and Quantitative Experiments, which uses heavy labeled peptide standards, and you can imagine that with small molecules, the peptide becomes a molecule with a collection precursor ions with adducts beneath it, just as peptides have a collection of precursor ions with charge and isotopic labelling applied to them.

Thanks so much!

Fuyan

I currently met a problem. I have some targeted compounds (e.g. m/z 1728.5, 1723.5). When I inserted Transition List with these compounds and checked for errors, the skyline said that "The precursor m/z 1728.5 is not measureable with your current instrument settings". But before this, I didn't import any results from my raw QE data. The transition list was the first thing I was editing before processing data with skyline. Is there any limited mass in the skyline setting?

Thanks,
Fuyan

I believe Skyline is telling you that 1728.5 is not within the minimum/maximum m/z that you can specify on the instrument settings in Skyline at:
Settings > Transition Settings > Instrument