1. In current versions of Skyline, the "Integrate All" setting really only affects the colors of the dots next to the Transitions in the Targets tree. If "Integrate All" is turned on, the dots next to the transitions will nearly always be green, unless the chromatogram peak really is completely flat. If "Integrate All" is turned off, the dots will be red if the apex of the peak for a particular transition is not close to the apexes of the other peptide transition peaks.
"Integrate All" should be turned on if you are doing quantification, and you care about the values of the peak areas.
"Integrate All" should be turned off if you are developing an SRM method and want to know which transitions have misshapen peaks.
Nowadays, it is very unlikely that "Integrate All" will have any impact on the numbers in your exported report, so it does not matter whether you have it on or off, but you should have it on in your case. (It used to be that when "Integrate All" was off, many of your transition peak areas would be NULL instead of their measured value, and that caused a big problem for people who forgot to turn it on when doing quantification, so, we fixed it so that it only affects the colored dots in the Targets tree).

2. I don't know the answer to this one.

3. Can you send us your Skyline document? In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.
If that .zip file is less than 50MB you can attach it to this support request. Otherwise, you can upload it here:
https://skyline.ms/files.url

I am not sure which columns in your report might be causing the report export to be slow. If I had to guess, I would say maybe if you remove the "Protein Abundance" column the report might go faster. It is not supposed to be slow for Skyline to calculate any of the columns in your report. If you send me your Skyline document I will try to improve the performance of Skyline on exporting that report.

4. I am not sure I understand your question. The way that peptides are grouped into proteins do not affect the peptide chromatograms or peak areas.

5. That's a good question. I do not know the answer.
I was going to say, if you have a .tsv file, you get it into the document with "File > Import > Assay Library".
You would use "Settings > Peptide Settings > Library > Build" if you have peptide search results. Here is a page which lists the types of peptide search results that you can build a library from:
https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BlibBuild
However, now that I look at that page, I see that it says you can build a spectral library from OpenSwath tsv, so now I don't know whether you should build a spectral library versus import an assay library.

6. I do not know the answer to this, but maybe someone else does.

7. The "Protein Abundance" column that you have in the report is the best way to sum the peak areas across all of the transitions under the protein. Those peak areas are normalized according to the Normalization Method that you have specified at "Settings > Peptide Settings > Quantification". If that Normalization Method is "None", then the Protein Abundance will usually be the same as the sum of all of the precursor's Total Area values. Sometimes the Protein Abundance value will be null if any of the transitions have missing or truncated peaks, because Skyline decides that that replicate's Protein Abundance value cannot be compared to the other replicates.

Please do send us your Skyline document. It is important that we figure out why your report export is slow, since that sounds like something that will be easy for us to fix.
-- Nick

Dear Nick,

Thanks for your reply.

The description of "Integrate All" is really detailed and helpful to me. I'll remember to turn it on when set settings at next time.

And for the export speed in question 3, it would be faster if I remove "Protein Abundance". In actual, this document contains ~300k precursors and would generate about 10M rows for export. If "Protein Abundance" was selected, it would take about one half day to export, while only several minutes without "Protein Abundance". Maybe in this case, the selection of precursors to calculate quantity for each protein would take much time? Meanwhile, the whole document would take about 800G so I didn't upload it now, but I can try if you want to have a look, please let me know if it would be useful.

The question 4 was to say if proteins would affect any step for searching and statistic control in mProphet. Because the library built with "Peptide settings -> Library -> Build" with a tsv file will have no protein included. I think it wouldn't but not sure before, thanks for your information.

For 5, I think the OpenSwath tsv in BlibBuild refers to search result from OSW and is used for visualization in Skyline (maybe?). But it truly worked with a tsv assay file so I tried it and got a blib with ion mobility annotated (though it would regard IM as drift time and need manually modify it to reduced IM).

And Thanks for your information about protein quantity in question 7. The "Protein Abundance" is removed now, so I directly aggregate precursors' quantities to protein after export. While normalization would affect quantification accuracy to some extent (in my case, with benchmark data). Maybe I need to try those different methods.

Thanks again for your enthusiastic help.

Best,
Ronghui

Thanks for Nick's help.

I have a new question about same (or similar) precursor m/z in library.

I noticed some peptides with same sequence and modifications, but different modification positions, would be dropped when adding them to document, becase of the match tolerance in settings. Then can I decrease this restriction to zero, to allow those peptides with same MS1 behavior but different MS2 information, for peptidoform detection? Do you think that's feasible in Skyline? We are going to test this for some software.

And does anyone have any suggestion about question 2 in the initial request? I'll be appreciate about that.

Best,
Ronghui

I do not understand what you are saying about mz match tolerance affecting whether Skyline gives you all of the positional isomers that you were expecting. Those two things are not supposed to affect each other.

There are two match tolerances in Skyline:
"Ion match tolerance" at "Settings > Transition Settings > Library".
When Skyline is looking at a spectrum in a spectral library, Skyline needs to figure out which theoretical transition (e.g. "y7++") corresponds to a particular m/z on the spectrum. The Library Ion Match Tolerance controls how much difference is allowed between the predicted and observed m/z's when interpreting spectral library spectra.

There is also the "Method match tolerance m/z". This setting affects whether Skyline thinks that a particular SRM chromatogram or MS2 spectrum in a raw file matches the m/z of a precursor in your Skyline document.

If you would like, you can send us your Skyline document and it might help us understand your question.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including spectral libraries and extracted chromatograms.
If that .zip file is less than 50MB you can attach it to this support request.
Otherwise you can upload it here:
https://skyline.ms/files.url

-- Nick