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Data Input and Output

Input

The main input for the NP³ MS Workflow1 (run command and major steps) are LC-MS/MS raw data files in mzXML or mzML formats (mzData format could also be used, but was not vastly tested). It fully supports the positive ([M+H]+) ion mode; and the negative ([M-H]-) ion mode may also be used, but the Steps 7 and 8 were not vastly tested for it and may lead to undesired results. The rest of the pipeline results can be used for the negative ion mode.

The NP³ pre-processed data and the clean data from the NP³ results are the inputs for the join_jobs command.

Equipment Resolution for Parameters Default Value

The default parameters values were refined using data acquired from a UHPLC-MS/MS-qTOF equipment, the UHPLC Acquity HClass Waters and the spectrometer ESI-QqTOF Impact II Bruker.

When using this workflow with data from other instruments, mainly the ones that have different resolutions, the tolerances and pre-processing filters should be reset accordingly.

Output

The workflow output is organized in folders and depending on the command can contain:

  • The pre-processed spectra in MGF files, containing all detected and filtered MS2 ion enriched with MS1 information. (Step 2)
  • The collection of consensus spectra from Step 3 and Step 5 in MGF files
  • The consensus spectra quantification results in CSV files, referred to as count tables. The count tables contains a consensus spectra ID, named msclusterID (representing a m/z and a retention time interval), by row and its quantification in each sample and indicators by column. The following count tables are created:
    • Clustering count tables (Step 4)
    • Clean count tables, with the clustering count table redundancies aggregated - remove fragmented clusters (Step 5)
    • Merged count tables (Step 8)
  • The count tables can also contain the following columns:
    • The libraries identification results (Step 6)
    • The ionization variants annotations grouped by type (Step 7)
    • The list of [M+H]+ representatives, suggesting the number of metabolites in the samples (Step 7)
    • The bioactivity correlation scores, that can be used to rank the consensus spectra. And the quantification grouping (Step 9)
  • The molecular networks (MN) of ionization variants annotations (IVAMN) and of spectral similarity (SSMN) files from Steps 7 and 10:
    • IVAMN (Step 7)
    • SSMN (Step 10)
    • SSMN filtered (Step 10)
    • IVAMN [M+H]+ (Step 10)
    • SSMN [M+H]+ filtered (Step 10)
  • The final reports with quantification, chemical and network statistics and analysis of the final results
    • A chemical space of the identified spectra using PCA

References

  1. C.F. Bazzano, et al. (2024). NP³ MS Workflow. Analytical Chemistry 96 DOI: 10.1021/acs.analchem.3c05829 ↩