Documentation
Parameters

Parameters

Parameters are critical for MS data processing. However, setting parameters are often challenging, especially for beginners. masscube provides a default parameter setting for users to start with.

The default parameters are optimized for most untargeted metabolomics studies. Users can also customize the parameters by providing a parameter file.

Key parameters

Here we summarized six most important parameters in masscube for untargeted metabolomics data processing:

  1. MS1 mass tolerance: the mass tolerance for MS1 feature detection. 0.005-0.01 Da is recommended.
  2. Intensity tolerance: the intensity tolerance for MS signals. 1000 is recommended for TOF data, and 30000 is recommended for Orbitrap data.
  3. Mass tolerance for alignement: the mass tolerance for feature alignment. 0.01-0.015 Da is recommended.
  4. RT tolerance for alignment: the RT tolerance for feature alignment. 0.1-0.3 min is recommended.
  5. MS/MS similarity score threshold: the threshold for MS/MS similarity score. 0.7-0.8 is recommended.
  6. MS/MS library: the MS/MS library for MS/MS annotation.

Default parameters

masscube automatically acquire the analytical metadata from the raw files including the mass spectrometer type and ionization mode. The corresponding default parameters will be applied based on the metadata.

Customize parameters

From the template, users can customize the parameters by providing a parameter file. The parameter file should be a .csv file with the following columns:

Parameter Value Explanation
rt_start 0 start of the analytical gradient; minute
rt_end 23 end of the analytical gradient; minute
ion_mode negative “positive” or “negative”
mz_tol_ms1 0.01 m/z tolerance for MS1 spectra; Da
mz_tol_ms2 0.015 m/z tolerance for MS2 spectra; Da
int_tol 1000 Intensity tolerance; 1000 for TOF and 30000 for orbitrap
align_mz_tol 0.01 m/z tolerance for alignment; Da
align_rt_tol 0.2 retention time tolerance for alignment; minute
msms_library path to the MS2 library; msp or pickle
ms2_sim_tol 0.7 MS2 similarity tolerance
run_normalization TRUE TRUE or FALSE; whether to run post-acquisition sample normalization
normalization_method pqn sample normalization algorithm
plot_bpc FALSE TRUE or FALSE; whether to plot base peak chromatogram for individual files
plot_ms2 TRUE TRUE or FALSE; whether to plot mirror plots for MS2 matching
run_statistics TRUE TRUE or FALSE; whether to run statistical analysis

More about parameters

Almost all parameters in masscube are tunable to ensure flexibility and adaptability for different datasets. For programmers, please refer to each function and object in the API documentation for more details.