diaPASEF Brett Phinney  2019-11-07

Hey Skyline team, I’m loading some diaPASEF into skyline. It’s super fast!!!

One question. How should I make the library? What formats/ search engines are you using? Does the library use the ion mobility?

I’ll be happy to share some data with everyone



Brendan MacLean responded:  2019-11-07

Thanks, Brett. Matt, Brian, and I worked hard on the performance.

For my recent processing, we have used MaxQuant msms.txt files for the libraries. When doing this, I think you want to try to disable its fragment ion charge state deconvolution, or you will end up with MS/MS spectra with charge 1 and 2 fragments merged into charge 1 making them less than ideal for subsequent DIA library use, though, not completely unusable. If you have other search tools you would like to try this with, ProteoWizard msConvert should make that possible with exported MGF or mzML. I have heard PEAKS also works. We would be interested in working with you to support any other pipeline you have, if the search provider is also willing to do the work to pass through the IM values.

We have also tested a Mascot pipeline that passes through IMS values, and, yes, they are important. Once you have your library built, open it with View > Spectral Libraries. Review some of the spectrum matches and make sure you have both RT: #.### and IM: #.###Vs/cm^2

Then you want to go into Peptide Settings > Prediction (sorry, we are going to move this to Transition Settings > Ion Mobility soon) and check "Use spectral library ion mobility values when present". In my most recent optimization analysis, I came away feeling that a "Resolving power" setting of 50 worked best for the data I have, but anyway from 20-50 should work okay.

Thanks for posting on the support board. Looking forward to hearing more as you get going with diaPASEF in Skyline.


Brett Phinney responded:  2019-11-08


Well I have made a little progress. I tried to use the most recent version of maxquant to search a timstof dda file and load it into skyline. Looks like it read the ion mobility data okay, but not the rt. See attached

I also tried to export a peaks skyline export, but the export file ballooned to 12GB after exporting for 12 hours and then my computer hard drive ran out of space... sigh.... Peaks seems to have an option to export a openMS file. Can I use that to build a library ?

thanks for all the help!!

Brendan MacLean responded:  2019-11-08

Strange. I have never seen us fail to get RT from MaxQuant before. Can you post the msms.txt file to https://skyline.ms/files.url ?

Someone here will have a look at it. Maybe check with the PEAKS team on the export to Skyline feature. We would be happy to help them work through the apparent bug in their implementation.

If you can export the OpenMS version and post that to our file drop point as well, we can have a look at whether it contains enough information for us to use in this case.

Thanks for pushing through the initial integration issues to help us work out the kinks with other software.


Brett Phinney responded:  2019-11-08

Thanks for the quick response!

put the files here


let me know if you need any more info or other files

matt.chambers42 responded:  2019-11-11

It looks like a MaxQuant Bug: all the values in the RT column are 0. In the OpenSwath CSV, all the "normalized" retention times are negative. I'm not sure if those 2 facts are related.

The OpenSwath CSV seems to have everything we need except filename. Or does OpenSwath create a separate CSV for each input RAW?