Custom spectral library from skyline

[cutleraging]

Hi Vadim and team,

I have generated a spectral library in skyline which I have exported as a report to be used in DIAnn. The reason for doing this is that I am analyzing histone PTMs and am interested in certain histone peptidoforms. Essentially this is an effort to reduce the search space. Therefore, I have set the proteins to be the peptidoforms, as I am interested in quantifying those. I have attached the report generated from skyline which I use as the input for DIAnn as well as the log which contains the command used to run DIAnn and the results. When identifying histone peptidoforms, it is quite important that the retention times be taken into account as well as the ion mobility (especially for isobaric forms). I have some questions that have come up when doing this analysis that I would appreciate your help on.

skyline spectral library.csv

report.log.txt

report.csv

1. When I search using this custom spectral library, I want to make sure that the retention times are being taken into account when searching for the histone peptidoforms. However, in the results, I can see that the identifications are not at the expected retention time (comparing to retention times in spectral library). I see that in the spectral library that DiaNN built, there are values in the Tr_recalibrated column. How can I ensure that DiaNN takes into account the RT in the spectral library? Is there a setting for an RT window or something like this?

2. Even though I include IonMobility column in the spectral library, in the spectral library that DiaNN built, all the values are 0. Why is this?

3. The LibraryIntensity that I provided in the spectral library was the MS/MS peak intensity of the corresponding product. I see these are all values between 0-1. Is this normal?

4. As I mentioned, I am interested in quantifying at the modified peptide level. In my spectral library, I provide the ModifiedPeptide and then in the ProteinName column, I provide a corresponding name for the modified peptide (e.g. H1.5-K33[un];K45[un];K51[un]). However, in the results, the Protein.Names column only contains a single name. How can I have it so that the Protein.Names column in the results corresponds to the ProteinName column in the spectral library? I want to do this so that I can use the values in the PG.Quantity column.

5. I have been varying the qvalue of my searches with the spectral library to see how this effects the results. (e.g. 50%). I find that when relaxing it, I get many more of the IDs that I expect, although at a cost. I notice that some of these new IDs are not at the expected retention time. Is there a way for me to determine the optimal qvalue for my situation? Also, what's the difference between precursor-level q-value and run-specific protein q-value?

6. Some of the histone peptidoforms are isobaric, and don't contain a unique fragment (although they do differ if looking at combinations of fragments). Should "No shared spectra" be enabled?

7. My samples are both label free single cells and bulk. Should the bulk samples be included in the searches with MBR enabled to boost the IDs in the single cells?

Thanks a lot! Ronnie