Table of Contents

Webinar 16: Small Molecule Research with Skyline
Questions and Answers

Webinar 16: Small Molecule Research with Skyline

Thanks to the 150 people who turned out to our November Skyline Tutorial Webinar entitled  Small Molecule Research with Skyline. Thanks too, to our guest presenter Will Thompson who provided a great tutorial involving creating a targeted quantification workflow from a small molecule transition list and small molecule method development. Our inhouse small molecule developer Brian Pratt also provided a good overview of how Skyline has evolved to supporting small molecules and where its headed in the future.

-- The Skyline Team


Will Thompson
(Small Molecule Researcher)


Presentation Slides

Tutorial Files
(28.6 MB)

Transition List

Brian Pratt
(Senior Skyline Developer)


Presentation Slides

Review Webinar 15

Large Scale DIA with Skyline
See video, presentation slides, and other files presented at the webinar.

Review Webinar 17

PRM Method Development and Data Analysis
See presentation slides and download data files presented at the webinar.

Other Skyline Resources

For more information on Skyline for small molecules visit the following links:

Questions and Answers

Q: If you have 2-3 transition for one molecule but you want to use only one transition for quantification, how do you do it?

Ans: In Skyline-daily 3.7.1.xxxx you will find a Transition.Quantitative field in the Document Grid view editor. If you add that to a view, it will enable you to set whether a peptide is quantitative or qualitative. Only the quantitative transitions will have their peak areas added during quantification operations.

Q: What is maximum number of datafiles you have processed in Skyline? Have you ever processed 1000 datafiles or more? 

Ans: We have never processed over 200 files in a single Skyline document during Skyline development, nor do we have any tests that do so. We have, however, processed DIA proteomics files with 1.5 million transitions over 125 runs. We see no reason a document with 1000 data files should fail, but if it does for anyone, we would love to take on the challenge of improving that. More likely the Skyline UI would get challenging to operate with this many files, and we might need suggestions on how that might best be presented. We do offer Panorama as an expandable repository for data processed in Skyline. The cases we know of that are processing more than 1000 runs generally process them in batches (say 96 runs) and then upload the batches to Panorama or some other data repository.

Q: Could you turn off certain views (bar graphs, etc.) if you need to for any reason?

Ans: Yes. The Skyline display is extremely customizable with the ability to open and close views, float them above the main window or dock them to it. Once you learn how to use the View menu and window docking, you should be able to achieve the display layout you desire.

Q: What is a double blank?

Ans: A double blank is a sample which contains neither the analyte nor its reference standard.

Q: Is there a way to predict product transition? Simillar to b and y ions?

Ans: This depends on the class of molecule you are targeting. Obviously, we are very good at this for peptides. We are working with collaborators on a tool called LipidCreater which seems to predict lipid fragmentation, and we have been told that it is also possible to do this for glycans. At the moment, neither of these are fully implemented in the Skyline software ecosystem. So, you are still really on your own with small molecules. Hopefully, the future will bring tools for this prediction where they are possible.

Q: Will CE be optimized using direct infusion of the standard? What's the advantage of this method?

Ans: We don't have any immediate plans to support CE optimization by direct infusion. The current method allows you to optimize potentially much larger sets of ions in parallel with one sample injection performed similarly to a normal sample injection during an experiment. No special prep, just a different instrument method. This has been extremely popular in proteomics.

Q: How can you export the columns for each of the tables you need?

Ans: Skyline has an extremely flexible and immediate reporting mechanism that allows you to chose from any of the elements in its internal data model from a view editor that lists them in a tree structure, with the ability to find them by name. To learn more, and you really should, because without this knowledge you are using a fraction of the power of Skyline, you can consult the Custom and Live Reports tutorial documentation.

Q: How do you get access the videos of previous webinars?

Ans: Recordings of all Skyline Tutorial Webinars are posted on the Skyline webite -- -- navigate to the Webinars link in the right hand column of the home page OR Webinars in the Get Started section in the middle of the page.  Either link will take you to an index of all the Skyline Tutorial Webinars and by clicking through to a specific webinar page you will see links for the presentation slides, supporting data, and a recording of the webinar (at the bottom), along with any related information to the webinar topic. 

Q: Do you have data sets we can walk through this seminar and practice?

Ans: The data will be posted on the webinar page around the time that the video recording is posted. These Tutorial Webinars are designed to make it possible for you to return to the video and follow along yourself (probably needing to click pause, as you go).

Q: To clarify: you imported all of the CE optimization runs and then executed the Skyline CE optimization functions at the end. It happened fast!

Ans: Yes, that is right. To perform the CE optimization 5 runs were necessary to keep the number of concurrent transitions at any time below 100. In the end those 5 runs were all imported into Skyline as a single "replicate" (i.e. single measurement of all the targets) and Skyline just stitched everything together to look like a single file to you. Though, using the Document Grid, you can still get access to which transitions were imported from which files. Remember you can always go back to the video to what a replay.

Q: Can Skyline provide the measured m/z from data?

Ans: Interesting question. It can in MS/MS spectra from spectral libraries, and when extracting XICs from HRMS data it will give mass errors in PPM, but we don't currently have a way for a user to access the m/z values that Skyline found in the raw data files, i.e. what the user entered in the method that the instrument ran. We do store that, so it wouldn't be difficult to expose through the Document Grid, but we haven't yet. Seems like a good idea. Currently, we tend to look at the raw data with SeeMS (another ProteoWizard tool) to understand what the mass spectrometer measured.

Q: Building the transitions (beginning of webinar) showed use of heavy H (I think shown as [M2H2] in the webinar). is there support for using 13C or 15N or are you limited to just typing in the mass?

Ans: As described during Brian Pratt's presentation, Skyline currently has extremely flexible adduct support. We believe it should support all types of labeling, but if you find a case where you are not satisfied, please give us the example to help us improve the software to meet your needs.

Q: Can Skyline analyze metabolomics data from QE instrument?

Ans: Yes. We hope to offer a small molecule HRMS tutorial webinar in 2018, but if you do some of the proteomics full-scan tutorials and combine them with the material in this webinar, perhaps you can figure it out before that webinar.

Q: Is it possible to specify a maximum tolerance range in addition to the explicit retention time in the transition list?

Ans: There is an Explicit Retention Time Window property, which you can also set. Currently, this only impacts retention time scheduling of the method, but does not limit the range Skyline will consider for peaks during raw data import. At the moment, if you measured the chromatogram, Skyline will consider it. It is possible we may implement more flexible support in the future. This is not the first request we have seen for limiting the retention time range within collected/extracted chromatograms.

Q: Can the mass tolerance be changed from Da to ppm?

Ans: We couldn't really come up with a case where that would be useful in SRM, but we definitely support PPM specifications of mass error tolerance when extracting chromatograms from centroided HRMS data. More on that for small molecules in a future webinar.











Dear Skyline Users,

Refining DIA workflows was a popular topic with over 180 attendees to our most recent Skyline Tutorial back in April -- Webinar #15: Optimizing Large Scale DIA with Skyline. We've posted data files and a recording for anyone wanting to review the content of the April session or its predecessor, Webinar #14: Large Scale DIA with Skyline.

We are very excited to announce our next webinar in the series:

Webinar #16: Small Molecule Research with Skyline

[registration closed]

When:  Tuesday, November 7, 8am (Pacific Time)
- plan for 1 hour presentation + 1/2 hour Q&A
  • Recent and upcoming advances in Skyline for small molecules
  • Targeted quantification workflow from wmall molecule transition lists
  • Small molecule targeted method development using Skyline

Emcee Brendan MacLean, Skyline Principal Developer will host this latest Skyline Tutorial Webinar and introduce the webinar's two presenters:  Brian Pratt, Skyline Senior Developer for Small Molecules will provide an overview of existing and future features of Skyline that support data analysis and visualization of "omics" research.  Then, Will Thompson, Assistant Director of the Duke Proteomics and Metabolomics Shared Resource,will present a tutorial for small molecule method development and optimization using Skyline. 

Join us, learn and help us to better meet your targeted "omics" research needs.

--Skyline Team


Brian Pratt

Brian Pratt (Senior Developer)

J. Will Thompson, Ph.D.

(Duke University)

Review Webinar 15

Optimizing Large Scale DIA with Skyline
See video, presentation slides, and other files presented at the webinar.