Data of MSX-DIA demultiplex error using MSconvert lxy0901  2020-03-31
 

Dear Skyline team,

I ran a DIA MSX experiment on QExactive HF-X set up using instructions by 5 m/z window, DIA, MSX 4.Data was collected in the profile mode. The method was set according to the template. I want do the demultiplex of RAW files by the Mscovert software, but this software displayed some error information. as shown:

Starting...
Opening file "J:\lxy\LXY-data\DIA\MSX\20191218\HFX_LXY_IgG_DIA_Methods_1_MSX_dia_4_5Da_20191218_F1_R1_20191219191652.raw" for read...
Calculating SHA1 checksum...
Processing...
Writing "J:\lxy\LXY-data\DIA\MSX\20191218\HFX_LXY_IgG_DIA_Methods_1_MSX_dia_4_5Da_20191218_F1_R1_20191219191652.mgf"...
Failed - System.Exception: SpectrumToIndices() Number of demultiplexing windows changed. Minimum window size or window boundary tolerance may be set too low.
在 pwiz.CLI.msdata.MSDataFile.write(MSData msd, String filename, WriteConfig config, IterationListenerRegistry iterationListenerRegistry)
在 MSConvertGUI.MainLogic.processFile(String filename, Config config, ReaderList readers, Map2 usedOutputFilenames) 在 MSConvertGUI.MainLogic.Go(Config config, Map2 usedOutputFilenames)

I want to know if there is a problem with my method settings?
Or can the data produced by the HF-X not be processed by this software?

My computer system is Window 7 service pack 1, 64 bit. The skyline software version is 64 bit 4.1.0.11717. The MScovert Version is 3.0.19161-f1b0b59b1.

The raw data and skyline files are already upload , "HFX_LXY_IgG_DIA_Methods_1_MSX_dia_4_5Da_20191218_F1_R1_20191219191652.rar"
And the problem and the setting is shown in the attachments.

Thanks.

Jessica Li

 
 
Mike MacCoss responded:  2020-03-31

Hi Jessica,
Thanks for your post. It is great to see that people are using the MSX-DIA data acquisition method!

Is it possible to see an example of your data files? From the error it looks like there might be an issue with the method that you are using. Also, it looks like you are generating an mgf file. I'm not sure if mgf file generation from an MSX-DIA file has been thoroughly tested. Some file formats have a bit of a problem supporting MSX demultiplexing because each scan number results in multiple demultiplex scans. In fact, even mzML had a bit of a problem with this initially.

Would you mind sharing a file? You can upload one here and just tell me the file name.
https://skyline.ms/files.url

You also mention using a method template. Would you mind sharing where that template was obtained? Also any screenshots showing the method setup would be great.

Cheers,
Mike

 
lxy0901 responded:  2020-03-31

HI Mike,

Thanks for your quickly reply. 

I have uploaded my data , file name is "HFX_LXY_IgG_DIA_Methods_1_MSX_dia_4_5Da_20191218_F1_R1_20191219191652.rar"

And the parameter setting is in the attachement.

I tried the MSX-DIA method the first time .  Look  forward to your kind reply.

Thanks!

Jessica
 
Mike MacCoss responded:  2020-04-08

Sorry for taking so long to get back to you. I have been distracted by some other things. I hope to get to this in the next couple of days.
-Mike

 
Tobi responded:  2020-04-09

Hello everyone,

just wanted to comment that you can at first just convert thermo raw data to mzmL or other formats without demultiplexing to have it in readable format with Notepad++. If this works and you find your isolation windows you have an indication that your files themselves are ok but the error might be occurring for other reasons like errors in window placement.

Mike, you know much more on MSConvert and such so please feel free to comment on this, also, what is your preferred file format for MSX and overlap DIA?

With best regards,
tobi

 
Mike MacCoss responded:  2020-04-09

Yeah there seems to be an error in the method where the demultiplexing algorithm can't solve the matrix. I just haven't had the time to figure out what the problem is yet or what to recommend to do differently.

Mike

 
Nick Shulman responded:  2020-04-09
Jessica,

The error is happening because your isolation windows overlap with each other. For instance, some of the isolation windows are:
865.1445-871.1445
870.14677-876.14677
875.14904-881.14904

Because of this, when msconvert sees the isolation window 870.14677-876.14677 it wants to try to decompose it into the three parts "870.146-871.144", "871.144-875.149" and "875.149-876.146".

It looks like there is a parameter called "minWindowSize" which you can pass to the demultiplex filter that will cause it to not consider the ignore the edge "870-871" and "875-876" windows and only look at the big middle one.

That is, you can run msconvert with the following commandline options:
msconvert.exe HFX_LXY_IgG_DIA_Methods_1_MSX_dia_4_5Da_20191218_F1_R1_20191219191652.raw --filter "demultiplex minWindowSize=2"

Then, msconvert can run without error. I am not sure yet whether it ends up producing a correctly demultiplexed file, since it has not finished running on my computer.
-- Nick
 
lxy0901 responded:  2020-04-10
Nick

Thank you for the method.

The raw data can be demultiplex now and the msconvert program can run completely on my computer.

I will try the application of the MSX method next.

Thanks!

Jessica