Hi Norelle,
There is not, as you are the first to ever request this. I think your only option would be to create two separate Skyline documents and analyze the Trypsin peptides separate from the Lys-N peptides. That means you would need to export two reports, combine them and do statistical analysis outside Skyline with a tool like MSstats, or your own R scripts.

I don't see anything blocking to analyzing the data quantitatively. It is just a bit less convenient. I will consider whether it would be easy to adapt what we have in Skyline to what you are requesting, and maybe it will be possible in the future.

Thanks for posting your feedback to the Skyline support board.

--Brendan

I would like to chime in to second this feature request. Being able to generate a spectral library from this type of combined digest would be very helpful.

Cheers

Dave

Hi Dave and Norelle,
Interesting point. Skyline would generate a spectral library from whatever your search tools output as the peptides it detected for your spectra. The enzyme settings in Skyline are completely irrelevant to this. It is all about how you set up your search engine. The library builder trusts the search results completely.

Then, if you set the enzyme in Skyline up as the "Trypsin and Lys-N" type of enzyme, Skyline will end up considering a super-set of the peptide cleavage that was actually possible. But, if you have your Peptide Settings - Library tab set up to only pick peptides that appear in the library, then you don't really care that Skyline believes combinations of Trypsin and Lys-N cleavage are possible, when actually only on or the other is possible, because Skyline will still only pick peptides that appear in the library, and only peptides that the search engine assigns to spectra will appear in the library.

So, I guess I think it may be more possible to achieve your desired result than I indicated the first time, as long as you have a peptide search engine that can deal with this enzyme combination correctly.

--Brendan

Hi Brendan,

I was thinking of a slightly different case from what Norelle originally described, where you might use multiple enzymes such as trypsin, AspN, GluC etc to maximize sequence coverage of a protein.

What you're describing would work, as long as you set the number of allowed missed cleavages high enough so that Skyline wouldn't miss possible peptides when it generates the peptide list. I'm thinking of a case where a long tryptic peptide might include one or more complete AspN peptides within the tryptic sequence. If you don't set the allowed missed cleavage sites to at least 2 (to encompass the AspN peptide within the longer tryptic sequence), then Skyline won't generate the correct list.

Thanks

Dave

This would be somewhat helpful for me as well; I have several peptides in a biological sample, digested with trypsin, that result from secretase cleavage of one protein of interest. Since they are semitryptic, technically speaking, Skyline will not recognize them. I've been toying with the notion of adding a custom secretase/trypsin enzyme definition, but I'm not sure if this is the best answer.

Cheryl

Hi Brendan,

As a follow-up question, what if I am doing "top-down"/peptidomics work? The search engine I am using has no enzyme specified. There for I should be able to enerate a spectral library in Skyline from whatever the search output can conduct MS1 filtering for quant. Yet as I proceed in the peptide search wizard for MS1 filtering, I come to a part where I have to specify and enzyme and "none" is not an option. If I chose trypsin for example, I have a no match error. Further, I cannot create a "none" option in Skyline.

Will there be a fix for this in the future?

Norelle

Hi Norelle,
The wizard is not really designed for that case, though you could define a "No Cleavage" enzyme yourself by setting the "Cleave C-terminal to" field to anything and then "Unless followed by" to all 20 amino acids "ARNDCQEGHILKMFPSTWYV". I just did this, and Skyline accepted it. This should entirely block cleavage and give you the whole proteins you are looking for.

Otherwise, you can drop out of the import wizard after the library is built, and then use View > Spectral Libraries to explore your newly created library and then the Add and Add All buttons to add peptide/whole-protein matches to your document.

There may be a fix for this in the future, but I hope these workarounds will suffice for now.

Thanks for posting your feedback.

--Brendan