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kevinkovalchik / RawTools

RawTools is an open-source and freely available package designed to perform scan data parsing and quantification, and quality control analysis of Thermo Orbitrap raw mass spectrometer files from data-dependent acquisition experiments.
Apache License 2.0
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Critical: Most of the parent ion info failed to be exported from Q Exactive Plus raw file #14

Closed microdou closed 5 years ago

microdou commented 5 years ago

Describe the bug Most of the parent ion info (MonoisotopicMass, ParentPeakArea, PeakFirstScan, PeakMaxScan, PeakLastScan, BaseLinePeakWidth(s), PeakParentScanIntensity, and PeakMaxIntensity) is missing when trying to export the _parse.txt (or _Matrix.txt in prior version) file from raw file.

Also note that ParentPeakFound field is largely identified as False.

Description of raw file MS2

Command line arguments mono tools/RawTools/RawTools.exe parse -f data/example.raw -p

Raw Files I've share the raw and exported files in Dropbox: https://www.dropbox.com/sh/x4dvsos376qjegj/AAAB0ZSdGBI8mraUcLRKZ_N7a?dl=0 The missing parent ion info can be easily identified in example.raw._parse.txt or example.raw._parse.csv

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Additional context

kevinkovalchik commented 5 years ago

Thanks for bringing up the issue. I'll have a look at the raw file this evening and see if I can get it fixed.

By chance, is monoisotopic precursor selection turned off in the QE instrument method? This causes the monoisotopic mass in the scan header to be 0 or incorrect, so RawTools can't find the peaks. If so you'll need to use the -R flag to refine the precursor mass. If this is the issue, it might be fixed in the next beta release. I've decided to identify precursor peaks using the trigger mass rather than the monoisotopic mass as it is generally higher in intensity.

Kevin

On Thu, Dec 6, 2018, 9:08 AM Junjie Wang <notifications@github.com wrote:

Describe the bug Most of the parent ion info (MonoisotopicMass, ParentPeakArea, PeakFirstScan, PeakMaxScan, PeakLastScan, BaseLinePeakWidth(s), PeakParentScanIntensity, and PeakMaxIntensity) is missing when trying to export the _parse.txt (or _Matrix.txt the old version) file from raw file.

Also note that ParentPeakFound field is largely identified as False.

Description of raw file MS2

Command line arguments mono tools/RawTools/RawTools.exe parse -f data/example.raw -p

Raw Files I've share the raw and exported files in Dropbox: https://www.dropbox.com/sh/x4dvsos376qjegj/AAAB0ZSdGBI8mraUcLRKZ_N7a?dl=0 The missing parent ion info can be easily identified in example.raw._parse.txt or example.raw._parse.csv

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  • OS: Arch Linux

Additional context This bug occurs when trying to export raw files of Q Exactive Plus, while Fusion Lumos raw files work fine. So I suppose it's an instrument specific bug.

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microdou commented 5 years ago

@kevinkovalchik Thanks for the prompt response!

  1. I'm not aware of the monoisotopic precursor selection switch and couldn't find it in QE method. What I can see from the method that might be relevant are:

    Full MS: Spectrum Data Type: Centroid
dd-MS2: Spectrum Data Type: Centroid
dd Settings: Exclude isotope: on

  2. As you can see, there are still several MS2 scans whose parent ions are detected by RawTools, so it's likely the "killswitch" was not turned on 😂

  3. Good to know there is an -R parameter, I will look into it.

  4. Yes, the trigger mass or most abundant monoisotopic mass with higher intensity may be more relevant in some quality control.

Thanks again!

microdou commented 5 years ago

@kevinkovalchik Tried the -R parameter, it didn't rescue the parent ion info... You may try playing with my raw file. Thanks!

microdou commented 5 years ago

@kevinkovalchik By the way, it may be better to export both the trigger mass and monoisotopic mass related info to a txt file, and let users to decide which to use, or just provide the options as parameters.

chrishuges commented 5 years ago

In QE methods, it MIPS is set under Peptide Match. It is not called MIPS

microdou commented 5 years ago

@chrishuges Oh yes, found it! Thank you! dd Settings: Peptide Match: -

Its options are: -, on, or preferred. I suppose - may be defaulted as off...

chrishuges commented 5 years ago

Yes the default is off. 'On' is the same as having MIPS enabled. 'Preferred' is a hybrid.

kevinkovalchik commented 5 years ago

1.4.0-beta.2 has been released, and should address this issue. The precursor peaks are now extracted based on the picked mass. I tested out your example file and it seems to go fine now! I agree that using the most intense peak in the isotope cluster would be nice, so this is something I will look into adding as an option.