Dear Skyline Users,

Our tenth Skyline Tutorial Webinar took on topic of Working with Modifications within Skyline. This prompted some great questions from attendees. Here are the answers:

Q: I am using stable isotope peptides as standards. I would like to create a spectral library of them in Skyline but I am having difficulties. I tried using the TPP but I could not figure out how to add the stable isotopes (I have some peptides with one heavy leucine but some regular leucine). I tried using Proteome Discoverer with dynamic modifications but Skyline did not recognize the modifications. Can you please discuss making a spectral library of stable isotope peptides in Skyline?

Ans: We can't help much with the TPP configuration issue, but they have an active support board. So, you might try posting to spctools-discuss@googlegroups.com. As for Proteome Discoverer, we do support importing search results from the tool in both .msf format (both pre- and post-v2.0) and exported as pepXML. If a specific file is not working as you would expect, you are encouraged to post to the Skyline support board (Help > Support from within Skyline). If you are willing to share your files, we can usually work out what is going wrong, whether it is configuration issue or a bug in Skyline. We'll do our best to get things working for you.

Q: How can we include immonium ions of modified residues if we want to follow those transitions?

Ans: While we understand that it would be nice to have more direct support for immonium ions in Skyline, it should now be possible to specify them through the Transition Settings - Filter tab, by specifying "Special ions", as was demonstrated for iTRAQ tag ions in the tutorial. This should allow you to target them in your experiments.

Q: For small molecules/metabolomics, are you planning to add common phase I and II metabolite modifications (eg methylation, sulphation, glucuronidation etc) on the modifications drop-list for MRM method development? That would be a great!

Ans: We are still working quite actively on improving Skyline small molecule support, and anything "That would be great!" is certainly a good candidate for inclusion in a future release. Thanks for the feedback.

Q: When will Skyline include its own database search engine and cut the umbilical cord from Mascot, Sequest etc. and other search tools?

Ans: We have no concrete plans at the moment to do this. Past experience has shown that researchers are rather attached to their peptide search pipelines, and it is not always easy to get them to discard their favorite for a more convenient one with which they are less familiar. That is part of why we took the approach of integrating with every known search pipeline that our users have requested, now over 20, we believe. Probably our most likely direct integration would be with the Pecan DIA search tool we have been developing in the lab.

Q: Is the auto-detection of modifications on search import only available in Skyline-daily?

Ans: No, this feature has been available since File > Import > Peptide Search was first released in Skyline v2.1, 2 years ago. Many of the improvements in the Skyline-daily release that was used for the tutorial are focused around the importing of "assay libraries" in the last section of the tutorial, which we didn't actually get to in the live webinar.

Q: When I import a Mascot search Skyline does not recognize all the modifications that are present in the search and so the spectral library isn't building properly. For example, in the search I may have up to ten differently modified versions of the peptide but only two go into the spectral library. Any idea on what the issue is?

Ans: This is the kind of question best answered through the Skyline support board (Help > Support from within Skyline). Often we will need to see the peptide search results you are working with to understand what is going on. If you run into problems like this, please do post to the support board, because there are probably others like you who won't post to the support board, and our only hope of improving the software is to hear about issues when they happen.

Q: If we have group of diagnostic ions in the MS/MS spectra, can we filter spectra by those diagnostic ions?

Ans: We would need a more detailed description of what you mean to be able to answer. You are encouraged to post this question to the Skyline support board with screenshots and more description. We'll do our best to help.

Q: Is it possible to set Skyline up to allow modification of both the n-terminal and the side chain of the first amino acid on a peptide?

Ans: At the moment, the only way of doing this is to define a single compound modification that encompasses the multiple modifications you would like to apply to a single site. Skyline does consider N-terminal and the N-terminal amino acid to be the same location (following Ron Beavis's lead from X! Tandem on this one). Here is a support request dealing with the issue:

https://skyline.gs.washington.edu/labkey/announcements/home/support/thread.view?rowId=23389

An example was given during the Q&A segment of the webinar, which is now posted for on-line viewing.

Q: What is the difference between Skyline and Skyline-daily? Why would you use the Skyline-daily in this tutorial?

Ans: Skyline-daily is the development or "beta" release of Skyline. It gets released as features are added to Skyline. It changes much more frequently than the main Skyline release. It has proven to be quite sable over the past 6 years, and some large labs, e.g. the CPTAC program that originally funded Skyline and the Aebersold lab have used Skyline-daily almost exclusively. Right after a public release, the two versions are nearly identical, but begin to diverge as time goes on. The current Skyline 3.1 release is now over 6 months old. In this tutorial, I wanted to use some improvements (mostly in assay library importing) that were implemented in the past 6 months.

Q: If there is time, could you give a brief overview of ion mobility filtering with Skyline?

Ans: Unfortunately, not for this webinar. Great topic for a future webinar. We are working very hard on ion mobility support with both Waters and Agilent and the Smith lab at PNNL.

Q: What about N-terminal modifications. I had asked this before but even if Nt-acetylation (very common) is defined as a modification and excluding n-terminal peptides is set to 0/ unchecked the many nt-acetylated peptides are not recognized as valid in skyline. I wonder why this is? (+42)

Ans: Sounds like another great post for the Skyline support board. Please post screenshots and a clear description of the problem, and we will do our best to understand and resolve the problem with you.

Q: How is the library for particular modifications built? By virtual simulation or with the real ions?

Ans: Unclear on what this is asking.

Q: How do you handle double modifications? For instance, phosphorylation and acetylation of the N-terminal residue?

Ans: This is answered above. You need to define a single compound modification for the double modification and apply that to the site that experiences the double modification. Double to you, but really just a single change in atomic composition, which can be expressed with the tools provided.

Q: Are you planning to add iTRAQ 8-plex modifications to Skyline?

Ans: Had never heard of this before the webinar. We will have a look, but as the support that allows the iTRAQ 4-plex definition in Skyline is completely customizable, anyone could add it, and share it using Skyline saved settings or just by passing around a Skyline document that contains the definition. When the document is opened, the definition will be added to the Transition Settings - Filter tab, "Special ions" list. Everything in the list could be removed or re-defined by you. These are just defaults for your convenience.

Q: Does this software support any chemical modification analysis? For example, chemical reactive compound modified protein analysis?

Ans: Would need more information to be able to answer this, but maybe that answers the question.

Q: Does Skyline support Glycosylations and Ubiqutinations?

Ans: Yes. The modification framework in Skyline is extremely flexible.

Q: I am using Idpicker 2.0 still. Does Skyline integrate with this. For IDPicker 3.0, the output can not be imported to skyline

Ans: We think we already support building libraries from the results of early IDPicker versions, by virtue of the fact that we worked so closely with the Vanderbilt lab that created it. We have been through several iterations of IDPicker file formats from idpXML/mzXML to mzIdentML/mzML. If you find for some reason that one of these formats does not work for you, we should be able to fix the code in Skyline that supports library building to work with your search results, because most of the code would already be there. It would likely be a very minor fix.