Question on Limits Tobi  2019-08-06

Dear Skyline team,

we have an outstanding project and want to work with DIA and Spectral libraries on a atrget list generated from huge fasta files in the GB range. Are there still any (fixed) limits for the number of peptides and transitions in the target list or the number of peptides in a spectral library that skyline can hendle? Older posts here yield different answers.

Best regards,

Nick Shulman responded:  2019-08-06
There are some limits when you do "Import Fasta", but the intention of those limits is to protect you in case your digestion or filter settings are going to result in an unexpectedly large Skyline document.

If you've told Skyline to only include peptides that are in your spectral library, then these limits do not apply. That is, if it says either "Library" or "Library and Filter" at "Settings > Peptide Settings > Library > Pick peptides matching" then Skyline does not limit the number of peptides or transitions that you get when you do "File > Import > FASTA".

The limits are 5 million transitions and 200,000 peptides.

The other time that this limit applies is if you have "Auto-select all matching peptides" selected at "Settings > Peptide Settings > Filter" and you do somethign which changes the set of peptides in your document.

These limits used to apply in more cases, but since that time we have added a Cancel button to most operations which might take a really long time and result in a Skyline document that is too large.
-- Nick
Tobi responded:  2019-08-07
Dear Nick,

thank you very much for the explanation.

In general i would find it always nice to have warning messages instead of fixed limits, especially with the new computational tools coming up which deal with a much higher number of PTMs.

Also, Skyline is extremely helpful to build the Input for Prosit. Its nice if the limit is lifted once a library is available, but in this context the limit might prevent building a large library in the first place.

Best regards,
Tobi responded:  2020-03-17
Hey everyone,

hope you are doing ok in these times and thank you for the recent patch.

I want to push this again since its an important topic. The issue comes when making libraries for more than 200k peptides with prosit because of the nonavailability to make respective input csv files. One solution would be to remove the limit in skyline or fix the fasta-to-prosit function in EncyclopeDIA (incorrect M(ox), range resets to 400-1000 mz, NCE for triply charged is higher than for doubly charged).

How do you deal with proteome scale predicted libraries, especially for MultiOrganism studies?

Best wishes,
searleb responded:  2020-03-17
Hi Tobi,
EncyclopeDIA is designed to make libraries for DIA, not for PRM/SRM. As a result:
1) it doesn't add in oxidized peptides, as they can get confused for more abundant unmodified peptides in wide window searches and inflate detection rates
2) it always builds fixed window libraries, e.g. from 400-1000 m/z. You can change these settings, but it's not designed to build a lot of smaller windows like 399.5-401.5 m/z, 548.2-550.2 m/z, 682.8-684.8 m/z, etc
3) it assumes that you have to fragment with a single fixed charge state (e.g. +3H), so it back-computes what the effective NCE would be for the off charge (+2H) using this formula:

However, if you post your use case on the EncyclopeDIA Issues Tracker ( then we can try to either build it in or work around it.
Nick Shulman responded:  2020-03-17

Are you getting an error message when you're trying to do something in Skyline? We can certainly fix that. What we typically do is make it so that instead of getting an error message you get that dialog with a progress bar and a cancel button.

Can you send me your Skyline document and the FASTA file that you were trying to import into it? (I'm assuming you ran into the error doing Import Fasta. If you ran into an error doing something else, then please send me the appropriate files).

-- Nick
Tobi responded:  2020-03-18
Dear all,

thank you very much for the responses. You find uploaded skyline and fasta as 200318_ImportError.rar

When importing both fasta the error states that the 200k peptide or max transition number were exceeded. It seems only doable with a library that already covers all those peptides to circumvent the limit. The dilemma is that even with Prosit you cant make the library as the fasta-to-csv with Skyline is affected by the limit in peptide number and in Encyclope DIA limited in mz-range and modifications.

Is there another way I'm not aware of? I will post to the EncyclopeDIA Issues Tracker as well to keep things in the right forum.

Best regards,