Dear Skyline developers,
Many thanks to your creative work in offering a better and better Skyline!
I want to use a single merged library to quantify several targets in PRM data from many samples. I searched the resquests&responses about merging libraries in Skyline, but I still have some doubts as follows:
1. Merging libraries from redundant libraries (.redundant.blib) and non- redundant libraries (.blib) both of which are built from .msf files (PD), or get a merged library directly from .msf files, what are the differences among the 3 merge libraries? According to my test, the merged libraries from .redundant.blib and .msf files are exactly the same. In addition, the 3 merged libraries contain the same identified peptides, but the size of the merged library from non- redundant libraries is smaller, which is due to redundant spectra are not included? generally, we can use the merged library directly from .msf files?
2. Since “Blibfilter refines the redundant library to choose just one representative spectrum for each peptide”, how to view the redundant spectra? If we activate or deactivate “keep redundant library”, how does it affect skyline results, e.g., for PRM data? The “best” spectra for all the identified peptides are exactly the same in the 3 merged libraries mentioned above?
3. During merging process, what are the detailed rules of selecting the “best” representative spectrum for each peptide, or give me a reference? Does it still use dotp value and the highest average similarity score during merging, the same as building a library? In my result, I found that Skyline picked a very noisy spectrum but with high intensity (see attached picture). Why does it not pick a much cleaner one with lower intensity? So intensity has priority in the picking process? Or is there an intensity threshold for Skyline to give priority to intensity? If yes, for all spectra with high enough intensity, Skyline will preferentially pick the one with less noise peaks and more targeted peaks?
4. In addition, does Skyline consider both MS1 and MS2 spectra quality in picking the “best” spectrum during merging? If yes, I want to limit it to only MS2 spectra quality for PRM analysis, how should I do?
5. Do the noise/interference peaks in the “best” spectrum have no influence in calculating dotp values in Skyline?
Thank you in advance!
Kind regards,
Antony |
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| Kaipo Tamura responded: |
2018-01-19 13:05 |
Hi Antony,
1. If I understand your question correctly, the libraries should be the same.
2. Keeping the redundant library does not affect Skyline results - only the filtered library is used.
3. When building a library in Skyline, the spectra that are selected are the ones with the best scores (as given in the input files). For your .msf files, this would mean the matches with the lowest q-values.
4. Only MS2.
5. Correct, these peaks are not used for calculating dotp values.
Thanks,
Kaipo |
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| user responded: |
2018-01-22 00:12 |
Dear Kaipo,
Thanks very much for your reply! Now it is much clearer to me.
Since we cannot explore the redundant spectra, in which cases does Skyline need the information in redundant libraries? Can we delete the redundant libraries because they occupy much space?
By the way, I have another question:
Can we use CiRT/SiRT to calibrate RT variations between a selected spectrum in the merged library and many different runs? In this case, the merge library should contain all the RT values of iRT peptides in different runs and automatically extract the corresponding iRT values for the selected “best” spectrum and the query spectrum. Does Skyline support this? If not, can we manually input iRT values somehow to make it work?
Kind regards,
Antony |
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| Kaipo Tamura responded: |
2018-01-25 15:51 |
Hi Antony,
Skyline does not use the information in redundant libraries, apart from filtering them to create non-redundant libraries. You can delete the redundant libraries (actually, Skyline will automatically delete them unless you have "Keep redundant library" checked on the Build Library dialog).
You can add iRT values from one spectral library to another spectral library by opening it as an iRT calculator, and clicking "Add..." -> "Add Spectral Library..." and selecting the other one.
Thanks,
Kaipo |
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Skyline picked the upper one.PNG