pep identification in DIA analysis

pep identification in DIA analysis heyang  2021-08-18


We just started to use Skyline to process DIA data. One questions is how we can export peptides confidently present in the data.


Nick Shulman responded:  2021-08-18
I think there are two different ways to do what you want.

One way is to use the relatively new "DIA peptide search" feature. To do that, go to:
File > Import > Peptide Search
and on the first page of the Import Peptide Search wizard, choose "DIA raw (deconvolute, search and build library)" for the "Start from" dropdown.
You can find the tutorial for this here:

A completely different way to go would be to add decoys to your document, extract chromatograms for both targets and decoys, and then train an mProphet model to score the identified peaks. For this, I would recommend looking at the advanced peak picking tutorial:

After you have done all of your DIA analysis, if you need to learn how to get lists of data out of Skyline, you should take a look at the Custom Reports tutorial:
-- Nick
heyang responded:  2021-08-18
Thank you, Nick.
Brendan MacLean responded:  2021-08-19
A better tutorial for learning DIA with an mProphet model is here:

And the associated webinars all use mProphet models which form the bulk of what the Skyline Team has published on this subject. The DIA-Umpire integration is relatively new and less well tested.
heyang responded:  2021-08-20
Hi Nick,

I tried to use DIA data directly to search database to build up library and data process. I got a problem, see attached. Could you help?

Nick Shulman responded:  2021-08-20
I do not know what could be causing this error. If you send us all of your files I can try to reproduce the error on my machine.

I believe the way that DIA Peptide Search works is by creating a .mz5 file which has fake spectra in it. The spectra are made by using the DIA Umpire algorithm to combine multiple spectra that all isolated the same precursor into different spectra that are easier for traditional peptide search engines to work on.

It looks like the problem that you are running into is that the .mz5 was not successfully created. I imagine this error is probably caused by some other error that happened earlier, and that other error is not being shown to you.

If you send me all of your files, I imagine the same error will happen for me.
I guess you would need to send me your .raw files, and your FASTA file. Also, please send me a screenshot of all of the pages of the Import Peptide Search wizard so I know what your settings are.

DIA peptide search is a relatively new feature in Skyline. It is important for us to figure out what is causing failures like this, so that we can either fix them, or at least change Skyline so that you are given enough information to know what went wrong.

-- Nick
heyang responded:  2021-08-24
Thanks, Nick. The raw data is huge and how should I send you data?

Nick Shulman responded:  2021-08-24
You can package all of your files into a .zip and upload it here:

I will also send you an email in case it's easier for you to use something like Google Drive or DropBox.
-- Nick
heyang responded:  2021-08-24
Hi Nick,

I wrapped raw data file and protein database and submitted them. thanks,

Nick Shulman responded:  2021-08-24
Thanks for sending those files.
I am working through the steps of doing a DIA peptide search on your WIFF file.
When you got to the "Configure Full Scan Settings" step of the wizard, what did you use as the isolation scheme? I was hoping that Skyline would let me use the isolation scheme called "Results Only" but Skyline gives me an error at the end of the wizard, so it must be I was supposed to choose something else. What did you choose?
-- Nick
heyang responded:  2021-08-24
Thanks, Nick and I selected "Results only" and fixed the margin problem (don't know why, 0.5 Da are missing in two rows). Heyi
Nick Shulman responded:  2021-08-24

I believe the error that you are getting is happening because the data needs to be centroided before Skyline sends it on to the DIA Umpire algorithm.

This seems to be a bug in Skyline: Skyline should be telling msconvert to centroid the data in addition to doing DIA Umpire stuff. MSConvert uses the term "peakPicking" instead of "centroiding".

I think you can work around this problem by first using MSConvert to create a .mzML with centroided data, and then using that .mzML file in Skyline instead of your .wiff file.
The commandline which I used to centroid your wiff file was:
msconvert.exe "20210818_123 VB UD2 360 0.165ugul 3ul C1.wiff" --filter "peakPicking true 1-"
msconvert.exe comes with ProteoWizard. You can install ProteoWizard here:

I am going to ask my coworkers and find out why DIA Umpire is choking on non-centroided data.

By the way, when you told Skyline to import the isolation scheme from your .wiff file, the reason that a few of the isolation windows did not get a margin was that the amount of overlap on the left and right sides were different.


The second window there, which goes from 897.5 to 904.5, overlaps by 1 unit on its left side (891.5-898.5) and 1.8 units on its right side (902.7-909.8). For this reason, Skyline was not able to find a margin that was appropriate for the left and right sides.

Thanks for letting us know about this DIA Umpire bug. I will ask around and find out if someone can fix it.
-- Nick

heyang responded:  2021-08-25

Thank you, Nick. Just let you know the error maybe related with our agency limit access on webpage. I can't download msconvert.ext due to limitation connection of our pc.


Matt Chambers responded:  2021-10-01

Skyline DIA-Umpire will do peak picking in the next Skyline-daily. Be aware though that peak picking on WIFFs is really slow so if you're going to run it multiple times it's better to convert them once to mz5 or mzML and run on those files instead.