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Crash when importing raw files erin weisenhorn  2022-05-25 14:18
 

I am running Skyline Daily with version 21.2.1.485 on Windows 10 and am trying to import MRM raw files from a Xevo G2-XS. When I import the files Skyline crashes and gives no error. I have replicated this on multiple computers. Under transition settings I have the mass analyzer set to 'TOF' and not centroided as another user reported causes error. I would really appreciate help with this and would be happy to provide the raw files.
 
 
erin weisenhorn responded:  2022-05-25 14:19

I will also add this is for small molecule analysis not peptides.
 
Nick Shulman responded:  2022-05-25 14:29
Can you send us your Skyline document and one or more of your .raw files?

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files such as spectral libraries or extracted chromatograms.

You should also zip up one or more of your Waters raw file folders and send them to us too.

Files which are less than 50MB can be attached to this support request.
You can upload larger files here:
https://skyline.ms/files.url

We have certainly had cases in the past where Skyline would spontaneously disappear because of a bug. What is supposed to happen is that the next time you start up Skyline, Skyline tells you that "Skyline had an unexpected error the last time you ran it." and ask you if you want to report the error. It sounds like that didn't happen for you, so the best thing for us to do is to try to reproduce this same bug on our computers.
-- Nick
 
erin weisenhorn responded:  2022-05-25 15:09
HI Nick,

The Skyline document and some raw files should be uploaded. I did get a few files to load, but the others have been consistently causing a crash. I haven't seen any errors upon restart. Thanks for your help.
 
Nick Shulman responded:  2022-05-25 15:24
Thank you for sending those files.
Yes, I have no trouble reproducing this error on my machine.
I will try to find someone who can fix this.
-- Nick
 
erin weisenhorn responded:  2022-05-26 13:21
Thank you, let me know if you need anything else from me.
 
warham responded:  2023-07-18 10:29
I'm having a similar challenge, Xevo g3 data, both mse and dda, crashes immediately. What is the work around?
 
Nick Shulman responded:  2023-07-18 12:26
warham,
We did fix Skyline so that instead of abruptly exiting you will see the "Unexpected Error" dialog.
If you are using Skyline version 22.2 or Skyline-daily and Skyline is still abruptly exiting, then it might be that this is a different bug, or it might be that we did not fix the original bug correctly.

The underlying error is caused by either a corrupted raw file, or a bug in the Waters software that we use to read raw files, or maybe a combination of both. I know we have told Waters developers about this error but I do not know the status of whether they have fixed it.

If you would like, you could send us your files too. The instructions would be the same as above:
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files such as spectral libraries or extracted chromatograms.

You should also zip up one or more of your Waters raw file folders and send them to us too.

Files which are less than 50MB can be attached to this support request.
You can upload larger files here:
https://skyline.ms/files.url
-- Nick
 
warham responded:  2023-07-18 18:31
Hi Nick,

I'm running

see attached

So I've tried it on two different installations, two different computers, running different flavors of Windows 11.

I've been talking with Waters too. I read here in the support docs, that as late as 2021 Brendan was still saying to use PLGS to process mse data for spectral library builds. I can't get Waters to send me Protein Lynx Global Server, in any form. I collected these data in DDA and MSE/DIA and I would sure like to build spectral libraries from my data.

I don't have PLGS to generate input for bibliobuild, the skyline DIA spectra library build from searching the .RAW expects you to have the SWATH sliding amu window data collection that I've used with Fusion Lumos's etc. but these data are MSe. I've tried the DDA data with the DDA search and spectral library build and it crashes as I've said. I have biopharmalynx and I can do the searches I need (mostly covalently modified proteins, ADCs etc) but while I can output from the biopharmalynx as a .csv, that I may end up converting into a .ssl, if I can get nothing else to work, and they also export .pkl files for the assigned spectra which might be a fit for skyline to be able to upload? but probably not.

So . . .

do you know where I might find a legacy version of PLGS, anywhere?
what is the format for the final_fragment.csv file that is the output from PLGS that skyline will use to build a spectral library?
I should be able to convert txt/csv version of the biopharma finder output into some .csv file.
does the skyline spectra library build also take .ssl format you describe in the tutorials?

I've been trying to make this work on my own now on and off for a few weeks. I could use some help.

Best,
Lance
 
Nick Shulman responded:  2023-07-18 19:33
Lance,

You should send us your .raw file and we will figure out why it is crashing.
You can package it in a .zip and upload it here:
https://skyline.ms/files.url

-- Nick
 
warham responded:  2023-07-19 06:50
I've uploaded a file 06132023-03, a DDA file which crashed on importing this morning, with default settings except no modifications.
 
Nick Shulman responded:  2023-07-19 09:14
Thank you for uploading that file.
I cannot get anything bad to happen with it.
Could you post some screenshots showing what you were doing in Skyline and what it looked like when things went bad?
-- Nick
 
warham responded:  2023-07-20 05:03
Hi Nick,

On my home computer this is the output from event viewer for the crash I ran this am:
 
warham responded:  2023-07-20 05:39
Hi Nick,

Ok, I had visual studio 2017 Pro installed. I installed visual studio 2022 community and reran the skyline search. The application event errors from event viewer are attached
 
Nick Shulman responded:  2023-07-20 08:23
Thank you for posting those screenshots of errors in Event Viewer.
I see that there was an AccessViolationException in "SpectrumList.spectrum" while MSAmanda was running.

Could you post some screenshots showing what you the pages of the Import Peptide Search looked like so I can see which raw files you asked Skyline to search and what settings you used?

I see that you also had an UnauthorizedAccessException inside of "ActivationContext.CreateFromName".
I have not seen such an error before, but, for an error like that we would recommend that you go to "Programs and Features" in Control Panel, uninstall Skyline, and then install Skyline again from the web site.

It might also be a good idea to try doing your peptide search in Skyline-daily instead of Skyline 22.2.
You can have both Skyline-daily and Skyline installed on your computer at the same time.
I do not think we have fixed any bug like this in Skyline-daily, but we might have. Knowing whether or not this error happens in Skyline-daily as well as Skyline 22.2 might be helpful in terms of figuring out what might be going wrong.
-- Nick
 
warham responded:  2023-07-20 09:01
Thank you Nick!

Ok, I used all default settings and the same DDA file I sent you yesterday 06132023-03.
I'll uninstall and reinstall skyline and try skyline daily this evening.

Thanks again,
Lance
 
warham responded:  2023-07-21 05:40
Hi Nick,

Here's a play by play upload fail.

Best,
Lance
 
warham responded:  2023-07-21 05:51
Hi Nick,

Here is the same series of captures after uninstalling and reinstalling skyline.
 
warham responded:  2023-07-21 06:03
Hi Nick,

Here's the error from importing into skyline daily. It's different, path finding error.
 
Nick Shulman responded:  2023-07-21 09:37
Lance,

Can you send me that FASTA file "trastuzumab.fasta"?
I don't think I will be able to reproduce the crash that you are seeing, so the best that we can do might be to send you the Skyline document that you would have gotten had the search succeeded.
-- Nick
 
warham responded:  2023-07-21 10:02
Hi Nick,

Here's the file. I can't see any issue with it. I hope you're able to figure out why mine is not working. Should all the files be on the C drive so the path calls assign correctly? I'll try that this evening. Thank you for our help and congrats on getting your implementation extending EncyclopeDIA released.

Best,
Lance
 
Nick Shulman responded:  2023-07-21 15:18

Lance,
Your FASTA file has only two proteins in it.
I don't think it's possible to do a successful MSAmanda search in Skyline with a FASTA file that is that small.
Part of the peptide search involves searching for decoys and figuring out a false discovery rate, and that just does not work unless there are a lot more potential peptides that could have been found.

This is the output that I end up getting:

Input files contain 32051 ms2 spectra
Searching file '06132023-03.raw'...
  Searching spectra 1 - 10000 of 32051 spectra
     Starting search against fwd database...
     Loading database
Loading database failed.
Could not find a part of the path 'C:\Users\nicksh\AppData\Local\Temp\~SK_MSAmanda\wpaglupq.vb1\AmandaDB\trastuzumab_map_TrypsinFULL_0_0_60\prot100000_digest.status'.
Search failed.
Search canceled.

One thing that you might want to do differently could be on the "Import FASTA" page of the Import Peptide Search wizard you check the checkbox that says "Import target proteins from a separate FASTA".
The first FASTA file that you tell Skyline about would be a big FASTA file representing the entire species in your sample.
The other FASTA file would be the one with the small number of proteins that you are actually interested in.
-- Nick
 
warham responded:  2023-07-23 05:15

Hi Nick,

I forgot to send this on Friday, it's the error in skyline daily that happens when importing MSe data through the encyclopeDIA file. I repeated it this weekend and got another error also attached.

The good news is that using the a proteome decoy file and my smaller file as the target worked for the DDA data. Thank you very much for that suggestion.

Follow-up question, is it possible to get the skyline-daily enyzme edit to properly include Nterm as well as Cterm cleavages as for enzymes like pepsin, see attached figures.

Thank you for all your help!
Lance
 
warham responded:  2023-07-23 05:26

Hi Nick,

Also, just to be clear, the DDA data import that works with the proteome and target FASTA files only works in skyline-daily. It does not work in the current skyline release.

You almost certainly new that, but just in case I didn't want to send imprecise information.

Best,
Lance
 
Nick Shulman responded:  2023-07-23 07:13
Lance,

I am not sure, but I believe that the EncyclopeDIA integration in Skyline-daily only works for a very specific workflow: you must have a narrow isolation window multi injection run which is used to build a chromatogram and a set of wide window single injection runs which you want to quantify.
For any other sort of workflow, you would either need to run EncyclopeDIA yourself directly to produce a .elib file, or you will need to wait until a future version of Skyline.

I am not sure I understand your question about pepsin. It looks like you are able to define that enzyme the way that you need to in the "Edit Enzyme" dialog. There is a separate question of whether an enzyme defined that way would work with the peptide search engines that Skyline runs, such as EncyclopeDIA or MSAmanda.
My understanding is that other search engines have their own ways of defining enzymes, and custom enzymes that you define in Skyline will probably not work with other search engines, but I do not know for sure.

If you are unable to get a peptide search engine to understand the enzyme that you want, a different thing that you could do would be to create a FASTA file with one entry for each possible peptide.
-- Nick