No enzyme option

support
No enzyme option Jason Held  2012-02-25
 
Hi Brendan,

I’ve got some data from samples that were digested with less-specific enzymes such as chymotrypsin, pepsin, thermoylsin etc that I’d like to do MS1 filtering analysis on and keep intact the amino acid numbering for each peptide in a protein. No enzyme is completely specific and i'd like to assess the reproducibility of peptide generation, and I think your MS1 platform is ideal for testing these less-specific enzymes with a targeted proteomics approach. For example, chymotrypsin cuts at far more than the 5-6 putative 'specific' cleavage sites that most people use under typical digestion conditions. At the end of the day, targeted proteomics is more about reproducibility than specificity.

I did my mascot searches with 'no enzyme' cleavage and would like to move all my peptides over to Skyline. However, I hadn't realized beforehand that there is no ‘no enzyme’ option in Skyline. I tried putting all amino acids cleavages sites, but then I’m limited to 9mers since I can only have 9 missed cleavages. I then tried making a background proteome and adding spectra from the library explorer, but that requires an enzyme to keep the amino acid info intact. I could make peptide lists and paste them in – however, this loses the amino acid # info and would be a nice feature to have. I'd suggest including a 'no enzyme' option in Skyline.

Cheers,
Jason
 
 
Brendan MacLean responded:  2012-02-25
Hi Jason,
You should be able to use the Library Explorer to add the peptides (without protein association). You don't need to paste peptide lists, but, yes, at the moment, there is no option to keep the protein association for unspecified cleavage. Searching for unspecified cleavage gets pretty expensive. There is currently an issue for supporting the case where you paste in the peptides and correct protein name into Edit > Insert > Peptides when you have a background proteome that uses specific cleavage.

https://skyline.gs.washington.edu/labkey/issues/home/issues/details.view?issueId=140

I can also imagine writing an algorithm to make this fast enough for importing a FASTA file and searching against library matches, but it would be moderately challenging. Maybe a good intern summer project or something.

At the moment, however, what we have is admittedly less than ideal for this case. As you note, it only allows peptide lists which lack the protein sequence and peptide location in that sequence.

It should still allow you to measure these peptides, though.

Thanks for your feedback.

--Brendan
 
1970530 responded:  2021-03-30
Dear Brendan,

We are facing the same problem as Jason some years before. Was there an update in the meantime or ist the solution you proposed in 2012 still the best way to handle the issue?

Many thanks for your help,

Lena
 
Brendan MacLean responded:  2021-03-30
Since the original post we have added support for semi-tryptic cleavage both as a protease definition (see "Trypsin (semi)" in Peptide Settings - Digestion tab) and for Background Proteome databases used for the Spectral Library Explorer - Add and Add All buttons with "Associate proteins" checked.

If you truly want to allow peptides with no defined protease cleavage, we still don't offer any additional support for that beyond adding your own named peptide lists through the Edit > Insert > Peptides (or similar, e.g. the Spectral Library Explorer - Add button) interface.

If this feels insufficient to you, would you mind explaining your experimental approach and how you are getting to where you feel you need a global mechanism to allow for peptides with undefined protease cleavage.

Thanks for continuing the conversation.

--Brendan