Hi Liti,
Can you post your files to:

https://skyline.ms/files.url

Also, it is always helpful to include exact details and/or screenshots. In this case the name of the protein you are using for the example of 200 v 100, and ideally a screenshot showing it in Skyline, or multiple screenshots in a PowerPoint slide deck.

The files we would need are the MaxQuant results you used to build your library and your Skyline file as a .sky.zip (using File > Share).

I think we should be able to give you a quick response once we have these. Sorry for the delay on a response.

--Brendan

Let me also add a bit more on the potential filters that get applied between your starting msms.txt file and what shows up in the Skyline Targets view:

• In the library build form, you are asked to supply a probability cut-off. Depending on the cut-off you choose, this may filter peptides listed in your msms.txt file.
• Skyline has a number of peptide-filtering settings that will further reduce the number of peptides appearing in the Targets view from what is stored in the spectral library (.blib file). e.g.
  • Missed cleavages - if you set this to a lower number than you used in your search (e.g. 0) than peptides with a higher number will be excluded
  • Filters: Min length, Max length, Exclude N-terminal AAs, etc. can all filter peptides between the library and the Targets view
  • Modifications: if you are missing modifications that you allowed MaxQuant to use, e.g. Oxidation (M), this would reduce the peptide count
  • Transition Settings: Filters - the precursor charges could cause peptides to be removed, the ion types and charges could combine with the Library settings to remove peptides
  • Transition Settings: Library - if you impose a "minimum product ions" limit, then peptides lacking enough matching transitions may be excluded
  • Transition Settings: Instrument - if the Max m/z value is set lower than the actual range of the data files, this can cause peptides to be excluded. This is important when taking a library from a DDA instrument to a triple-quad SRM instrument, because you don't want Skyline suggesting precursors that the triple-quad cannot measure.

I am sure there are others, but I wanted to point out these filters exist and often need to be tweaked to get everything you want. This has come up before, and I continue to consider if there are better ways for us to handle DDA and DIA search tools when a user wants to use Skyline target everything that was found by an upstream tool, as you seem to want to do.

Dear Brendan,
Thank you for the tips, in fact, I was losing information because of these settings. Mainly because of the missed cleavage numbers. It was set to zero.
I changed it according to the msms.txt result and I have more peptides now.
I will work on this and see if now I have what I need for my analysis.
I come back here if I still have questions.
Thank you again.

Liti

Dear Brendan,
I'm working on my DIA results and a question came up that I'm not sure about.
Considering that all my DDA (library) and DIA acquisitions were in the same instrument and with the same setting e.g. time and gradient for peptides elution, Does the retention times in my DIA results must match with the library data?
I'm asking because I noted that it is not all the peptides that have the best peaks matching with the retention times from the DDA data that the library was created.
I'm wondering if this is important or not and if I need to consider only the DIA peaks in the same retention time from my library.

Thank you again for your help,

All the best,

Liti

Hi Liti,
You have two options for how you might translate the retention time information in your DDA library for use with your DIA data:

1. Ask Skyline to use the raw times in the library, which may be appropriate in the situation you have described where you maintain the same instrument and the same chromatography. This method is covered in both the Basic DIA tutorial (https://skyline.ms/tutorial_dia.url) and the original Skyline Tutorial Webinar on DIA (https://skyline.ms/webinar02.url). The key is to choose the option "Use only scans within X minutes of MS/MS IDs" in the Transition Settings - Full Scan tab.
2. Use an iRT retention time predictor built based on your DDA library. This option is covered in the Analysis of DIA/SWATH Data tutorial (https://skyline.ms/tutorial_dia_swath.url) and a more recent Skyline Tutorial Webinar on the subject (https://skyline.ms/webinar18.url). Here you build an iRT table into your library and then choose "Use only scans within X minutes of predicted RT". Skyline can even build the iRT table using endogenous peptides in your samples as the anchor peptides (or iRT standards) if you have not add an RT standard mix to your samples.

Hope these resources help get you what you are looking for.

--Brendan