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MS converter issue - Most urgent - need help

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MS converter issue - Most urgent - need help amey shirolkar  2022-05-02
 
Hello Team Skyline,

For my firstever DIA experiment I am attempting to convert my .raw files into .mzML files so as to upload them into Skyline for analysis, but I am getting following error message for past 4 days.

Please help its very crucial for me.
Regards.

- Amey Shirolkar, PhD
Scientist
Enveda Biosciences, Visakhapatnam
amey.shirolkar@envedabio.com
 
 
Nick Shulman responded:  2022-05-02
It sounds like you have asked MsConvert to demultiplex your overlapping DIA windows.
The error you are getting only happens if you have told added a "Demultiplex" filter in MSConvert.
I believe that error happens if there is a little bit of extra margin in the way that windows overlap with each other. The way to deal with this is to tell MSConvert that there is a minimum window size, and the little bit of margin window overlap is not supposed to be a separate demultiplexed window.

It is not possible to specify a minimum window size using the MSConvert Graphical User Interface that you are using. You need to use the commandline version of msconvert.exe.

There is some information about how to specify the minimum window size at the bottom of this support request:
https://skyline.ms/announcements/home/support/thread.view?rowId=50555

If you would like, you can send us your .raw file and we can tell you more about what is going on.
You can upload files here:
https://skyline.ms/files.url

-- Nick
 
Nick Shulman responded:  2022-05-04

Thank you for uploading your raw files.
It looks like the problem is that only some of your isolation windows are overlapping with other isolation windows.
That is, the isolation windows from [395-405] all the way up to [570-580] all overlap with their neighbors.
However, after that, the isolation windows from [570-580] to [900-910] do not overlap with their neighbors.
If you want to use msconvert to demultiplex this data, you are going to need to create two separate .mzML files with the two different types of scans in them, and then use msconvert again to merge those two mzML files into one mzML file.

So, the commands I would run are:

  1. Tell msconvert to create a file called "overlapped.mzML" containing the demultiplexed overlapping MS2 spectra
    msconvert.exe CVL31974_DIA.raw --filter peakPicking --filter "mzPrecursors [400.43188,405.43417,410.43646,415.43872,420.44101,425.44327,430.44553,435.44781,440.4501,445.45236,450.45465,455.45691,460.4592,465.46146,470.46375,475.466,480.46829,485.47055,490.47284,495.4751,500.47739,505.47964,510.48193,515.48419,520.48645,525.48877,530.49103,535.49329,540.49554,545.49786,550.50012,555.50238,560.50464,565.50696,570.50922,575.51147]" --filter demultiplex --outfile overlapped.mzML
  2. Tell msconvert to create a file called "regular.mzML" which contains the MS1 spectra and the non-overlapping MS2 spectra
    msconvert.exe --outfile regular.mzML --filter peakPicking --filter "mzPrecursors [0,575.51147,585.51605,595.52057,605.52515,615.52966,625.53424,635.53876,645.54333,655.54785,665.55243,675.55695,685.56152,695.56604,705.57062,715.57513,725.57971,735.58423,745.58881,755.59332,765.5979,775.60242,785.60699,795.61151,805.61609,815.62061,825.62518,835.6297,845.63428,855.63879,865.64337,875.64789,885.65247,895.65698,905.66156]" CVL31974_DIA.raw
  3. Tell msconvert to create a file called "merged.mzML" which contains the everything from "overlapped.mzML" and "regular.mzML":
    msconvert.exe --outfile merged.mzML --merge --filter sortByScanTime overlapped.mzML regular.mzML

-- Nick