Gas-phase-fractionation and iRT

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Gas-phase-fractionation and iRT Joerg  2022-11-18
 

Hi,
I have a problem with iRT alignment with my gas-phase fractionation-SWATH-runs. As I am doing GPF for the SWATH-analyses some of the iRT peptides (Pierce C18) are fragmented only in fraction while others are fragmented in fraction 2 and so on, but none of the peptides are fragmented in the different gas-phase-fractions. I have full-MS-spectra for the entire precursor region in every run, though. Is there a possibility to align the runs according to the precursor signals or do I have to fragment each peptide in each run?
Many thanks!
Joerg

 
 
Nick Shulman responded:  2022-11-18
Skyline does not have any sort of feature to tell it "these runs have retention times that are so similar that they can all use the same iRT alignment".

Someone had this question a few years ago:
https://skyline.ms/announcements/home/support/thread.view?rowId=45416

I told them to use msconvert.exe to combine all of the separate .raw files into a single big .mzML file with the command line arguments:
--merge --filter sortByScanTime
and that seemed to work well for that user.

-- Nick
 
Joerg responded:  2022-11-18
Hi ick,
thanks for the quick help! Should this also work for .wiff files?
Thanks,
Joerg
 
Brian Pratt responded:  2022-11-18
Yes, in both cases you end up with a single .mzML file that you use with Skyline instead of the original unconverted files.
 
Joerg responded:  2022-11-27
Hi,
thanks for the recommendations! Although it took quite long (~3 h per sample) the combination of the GPF-fractions worked out. However, now I get the following error message after a couple of hours (processing the first mzmL-file):

[221111_Ullrich_220697 (1 of 30)] [step 7 of 7] processing DIA window: 1/120
Error occurred running process.
Warning: vendor peakPicking was requested, but is unavailable for this input data. Using ProteoWizard centroiding algorithm instead.
High-quality peak-picking can be enabled using the cwt flag.

Command-line: C:\Users\Admin\AppData\Local\Apps\2.0\BPQO6278.1CX\2GT6ZBTD.GHP\skyl..tion_e4141a2a22107248_0016.0002_c984fd7cba69179e\msconvert -v --32 -z --mz5 -o "C:\Users\Admin\AppData\Local\Temp" --outfile c0fmroa3.ohp.mz5 --acceptZeroLengthSpectra --simAsSpectra --combineIonMobilitySpectra --filter "peakPicking true 1-" --filter "diaUmpire params=C:\Users\Admin\AppData\Local\Temp\pkpnb3z4.ufw.params" "D:\2022\Proteomics\221111_Ullrich_220697ff\mzmldata\221111_Ullrich_220697.mzML"
Working directory:
Conversion failed.

Is there anything I could change? Otherwise, I would try to process the different GPF-fractions each with a subset of the retention time standard and combine the results later?! Or do the RT-alignment solely on the Full-MS-level, if possible?

Many thanks for the great support!
Best Regards
Jörg
 
Nick Shulman responded:  2022-11-27
When you first created the mzML file, I think you should have included the following extra arguments at the begining:
 --filter "peakPicking true 1-"

The problem is that centroiding cannot usually be performed on mzML files, so, if the data is going to need to be centroided, you need to include that filter argument when the raw (or wiff) file is first converted to mzML.
--Nick
 
Joerg responded:  2022-11-27
Many thanks,
I will give it a try!
Best
Joerg
 
Joerg responded:  2022-11-27
Hi,
sorry to bother you again, but using this argument msconvert ends with an error:

[SpectrumList_PeakPicker] Warning: vendor peakPicking requested, but peakPicking is not the first filter. Since the vendor DLLs can only operate directly on raw data, this filter will likely not have any effect.
Warning: vendor peakPicking was requested, but is unavailable for this input data. Using ProteoWizard centroiding algorithm instead.
High-quality peak-picking can be enabled using the cwt flag.
writing output file: .\221111_Ullrich_220697.mzML
[msconvert] Caught unknown exception.
Please report this error to support@proteowizard.org.
Attach the command output and this version information in your report:

ProteoWizard release: 3.0.21112 (b41ef0ad4)
Build date: Apr 22 2021 03:26:13

Should I contact the msconvert-dashboard for this or is there any workaround?
Best Regards
Joerg
 
Nick Shulman responded:  2022-11-27
What was the whole commandline that you gave to "msconvert.exe"?

The argument:
--filter "peakPicking..."
must appear before all of the other "--filter" arguments that might be on the commandline.

If you tell us what your whole commandline was, we can tell you how you need to reorder the arguments so that they work.
-- Nick
 
Joerg responded:  2022-11-28
Oh,
that was in the wrong order then. I now cahnged it to:

msconvert --filter "peakPicking true 1-" --merge --filter sortByScanTime 221111_Ullrich_220697_1.wiff 221111_Ullrich_220697_2.wiff

I now get the following Warning and unfortunately also an "unknown exception".

Something still doesn't work, sorry...
Joerg



Warning: vendor peakPicking was requested, but is unavailable for this input data. Using ProteoWizard centroiding algorithm instead.
High-quality peak-picking can be enabled using the cwt flag.
[msconvert] Caught unknown exception.
Please report this error to support@proteowizard.org.
Attach the command output and this version information in your report:

ProteoWizard release: 3.0.21112 (b41ef0ad4)
Build date: Apr 22 2021 03:26:13
 
Nick Shulman responded:  2022-11-28
Can you send me those two .wiff files and the .wiff.scan files that go along with them? I will figure out why that "Caught unknown exception" is happening.

If those wiff and wiff.scan files are less than 50MB you can attach them to this support request. You can upload larger files here:
https://skyline.ms/files.url

I am not sure why you would be getting the warning "vendor peakPicking was requested, but is unavailable for this input data".
It is telling you that things might go better if instead of:
--filter "peakPicking true 1-"
you changed that to:
--filter "peakPicking cwt"

It is possible that making that change might make it so that you are able to successfully convert your centroided mzML files, but, even if the workaround does work it would still be helpful if you could send us your .wiff and .wiff.scan files because I think we will still need to fix whatever is causing the unknown exception.
-- Nick
 
Joerg responded:  2022-11-28
Many thanks,
I uploaded a zip-file containing the two runs (221111_Ullrich_220697.zip).
I will try the --ctw option in the meantime!
Best
Joerg
 
Nick Shulman responded:  2022-11-28
Thanks for sending those files. MsConvert is still chugging away on my computer converting those files to mzML. I have not seen the "vendor peakpicking unavailable" warning. Maybe I will see a warning like that if I let this thing run all the way to the end, but I would have expected to see that warning sooner.

I see that you are using a pretty old version of MSConvert from April 2021.

I would recommend that you download the latest ProteoWizard from the ProteoWizard page:
https://proteowizard.sourceforge.io/download.html

You will probably also want to uninstall older versions of ProteoWizard from your computer so that you do not get confused by accidentally running an older version.
-- Nick
 
Nick Shulman responded:  2022-11-28
msconvert.exe is choking on spectrum number 237646 in 221111_Ullrich_220697_1.wiff
That spectrum seems to have zero m/z's and intensities in it. I am not sure whether that means the .wiff file is corrupted, or whether this is a bug in msconvert.exe.

You can work around this problem by telling msconvert to skip over that particular spectrum.
You would invoke msconvert three times: once for each of the .wiff files to create a centroided mzML file, and then a third time to combine the two mzML files into one:
msconvert.exe --filter peakPicking --filter "index 1-237645 237647-" 221111_Ullrich_220697_1.wiff
msconvert.exe --filter peakPicking 221111_Ullrich_220697_2.wiff
msconvert.exe --outfile myMergedFile.mzML --merge --filter sortByScanTime 221111_Ullrich_220697_1.mzML 221111_Ullrich_220697_2.mzML

(I am running these commands on my computer now, so I am not sure whether it will complete successfully. If it turns out that I made a mistake with the command line arguments I will edit this post)
-- Nick