Scan type HCD not recognized

[JB91451]

Dear all,

in order to use the very same peaklist files for different search engines, I had to convert some of my files to mzXML before searching them. When I use these files with MSFragger 3.5 I get a warning message during file checking and calibration saying: "Activation type HCD not recognized, activation filtering may be inconsistent! Please contact the developers to fix and/or disable activation type filtering" The files originate from a QExactive plus measurement in DDA mode and were converted via msconvert. Within the mzXML files, msLevel=2 scans have a tag 'activationMethod="HCD"' along with the precursor information. Also, the number of scans displayed during parameter optimization for each file seems valid. Therefore I would like to ask, whether it is save to ignore this warning (I have not mixed activation schemes etc.)?

Best regards, Juergen

Please find the used parameter file here. 'activation_types = HCD' had to be specified as without this information the searches immediately stopped.: PPutida_Proteogenomics_semiLysC_MSFragger.params.txt

Below is an example for a MS2 scan in the mzXML files:

<scan num="3702"
      scanType="Full"
      centroided="1"
      msLevel="2"
      peaksCount="366"
      polarity="+"
      retentionTime="PT1688.78S"
      collisionEnergy="27.5"
      lowMz="100.076606750488"
      highMz="1009.432556152344"
      basePeakMz="120.0816037"
      basePeakIntensity="5.7522012e05"
      totIonCurrent="2.891132e06"
      msInstrumentID="1">
  <precursorMz precursorScanNum="3700" precursorIntensity="8.67563701931e05" precursorCharge="3" activationMethod="HCD" windowWideness="3.0">334.5353245056</precursorMz>
  <peaks compressionType="none"
         compressedLen="0"
         precision="64"
         byteOrder="network"
         contentType="m/z-int">QFkE5yAAAABAjU1iwAAAA...</peaks>
</scan>

[fcyu]

Hi Juergen,

Can you send on of the mzXML file to us?

Thanks,

Fengchao

[JB91451]

Hi Fengchao,

if you would open me a box folder, I can try to do so. However, the files are really large (8-12 Gb).

[fcyu]

Sure, here it is: https://www.dropbox.com/request/WG7nJrF13dPr22XpthE4

Best,

Fengchao

[JB91451]

Thanks, it should upload. I also added the mzbin, search results (which seem good to me) and the fasta.

Best, Juergen

[fcyu]

Hi Juergen,

FYI, the dropbox folder is still empty. Are you still uploading the files?

Best,

Fengchao

[JB91451]

Hi Fengchao,

thank you for informing me. There was a web-connection error with our server and thus the upload failed. Meanwhile it should be finished as I repeated it and got a confirmation.

Best, Juergen

[fcyu]

Hi Juergen,

Thanks for your files. I couldn't reproduce the warning message. Everything looks good from my test. Attached please find the log file.

Best,

Fengchao log.txt

[JB91451]

Dear Fengchao,

Thank you for checking this. I still get the same error but as the number of spectra is the same as in your log file I guess I can simply ignore it?!

Best, Juergen

[fcyu]

I don't know if it is safe to ignore it. I think you need to troubleshoot from your side.

Best,

Fengchao

[dpolasky]

Hi Juergen, If you remove the activation_types=HCD parameter from the MSFragger parameters file (or change it to activation_types=all), the message should go away. Unless you need to filter in a file with multiple activation types, this parameter shouldn't be needed, and any unexpected behavior from trying filtering an mzXML file can be avoided. Best, Dan

[JB91451]

Thank you both for your recommendations. I will try to run it with activation_types=all (I added the HCD as leaving this parameter blank did not work at the beginning) and try in parallel with the original raw files. However, I am quite optimistic that the results will be identical or close.

Thanks again, Juergen

[fcyu]

OK, I see the reason.

Dan, does it mean that we won't be able to support mzXML in the future? At least the activation_types parameter won't support it.

Best,

Fengchao

[dpolasky]

Yes for activation type filtering we probably won't support mzXML (unless there's a lot of demand for it), but can change it so that it doesn't try to filter rather than giving this scary error message

[fcyu]

Actually, the mzXML format has the activation method information:

<precursorMz precursorScanNum="839" precursorIntensity="3.544250343306e06" precursorCharge="2" activationMethod="HCD" windowWideness="3.0">324.172088534973</precursorMz>

I guess we can do something to recognize it.

Best,

Fengchao

[dpolasky]

yes the info is there in mzXML so we could support it, just need to test to find all the different ways it can be reported by different instruments/vendors

[fcyu]

I tested it with activation_types = HCD. MSFragger actually can recognize the activation type from mzXML format. And there are still no error messages. Then, I am not sure why @JB91451 got it,

Best,

Fengchao

[JB91451]

Dear all,

I tried with activation_types = all over night and there is no warning anymore. As the files are identical to the ones produced with HCD filtering I think it was safe in my case to ignore the warning (at least in my case). Nonetheless I will use the files created without filtering to be sure.

Many thanks again, Best, Juergen