Comet Search Error (Code 256) with TimsTof Flex MALDI Data

[Yang Qianying]

Dear Jimmy Eng,

I encountered an error (Code 256) while using Comet for database searching. Below are the details of my setup and troubleshooting attempts:

• Instrument: TimsTof Flex, MALDI-2

• Sample Preparation: 384-well AnchorChip

• Acquisition Mode: MS1-only (no MS2)

• Data Processing:

  • I tested two different types of .d files:

    1. Combined scan: All 384 wells merged into a single .d file (with and without Tims).

    2. Single well scan: A .d file from an individual well (with Tims).

  • All searches resulted in error Code 256.

• Data Conversion:

  • Used MSConvertGUI to convert .d files to mzXML.

  • Applied high-high parameters downloaded from the Comet website.

  • Also tested modified parameters with adjusted mass range (attached).

I have attached a screenshot of the error message and my parameter files for reference.
Could you please help me identify what might be causing this issue?

Thank you for your time and support!

Best regards,
Qianying Yang 

[Jimmy Eng]

Hi. Comet identifies peptides by comparing tandem mass spectra (or MS3 spectra) against sequences in the database.  The "ms_level = 2" parameter setting specifies which spectra to analyze, in this case MS2 spectra.  Valid values for this search parameter are "2" for MS2 spectra or "3" for MS3 spectra .  Because you have no MS2 spectra to analyze in the files that you are searching, there is nothing for Comet to do.  That's the reason for the message "Load spectra: warning - no spectra searched".  The solution is to search files that contain MS2 spectra.  Good luck.

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