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:
- MS1 mass tolerance: the mass tolerance for MS1 feature detection.
0.005-0.01Da is recommended. - Intensity tolerance: the intensity tolerance for MS signals.
1000is recommended for TOF data, and30000is recommended for Orbitrap data. - Mass tolerance for alignement: the mass tolerance for feature alignment.
0.01-0.015Da is recommended. - RT tolerance for alignment: the RT tolerance for feature alignment.
0.1-0.3min is recommended. - MS/MS similarity score threshold: the threshold for MS/MS similarity score.
0.7-0.8is recommended. - 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, you can customize the parameters by providing a parameter file. The parameter file should be a .csv file with the following columns:
| Parameter | Value | Explanation |
|---|---|---|
| ion_mode | positive | MS ion mode, “positive” or “negative” |
| ms_type | qtof | type of MS, “orbitrap”, “qtof”, “tripletof” or “others” |
| is_centroid | yes | whether the raw data is centroid data, “yes” or “no” |
| mz_lower_limit | 0 | lower limit of m/z in Da |
| mz_upper_limit | 100000 | upper limit of m/z in Da |
| rt_lower_limit | 0 | lower limit of retention time in minutes |
| rt_upper_limit | 10000 | upper limit of retention time in minutes |
| ms1_abs_int_tol | 1000 | absolute intensity threshold for MS1, recommend 30000 for Orbitrap and 1000 for QTOF |
| ms2_abs_int_tol | 500 | absolute intensity threshold for MS2, recommend 10000 for Orbitrap and 500 for QTOF |
| mz_tol_ms1 | 0.01 | m/z tolerance for MS1, default is 0.01 |
| mz_tol_ms2 | 0.015 | m/z tolerance for MS2, default is 0.015 |
| scan_scan_cor_tol | 0.7 | scan-to-scan correlation tolerance for feature grouping, default is 0.7 |
| mz_tol_alignment | 0.01 | m/z tolerance for alignment, default is 0.01 |
| rt_tol_alignment | 0.2 | RT tolerance for alignment, default is 0.2 |
| correct_rt | yes | whether to perform RT correction |
| merge_features | yes | whether to clean feature table by merging features with almost the same m/z and RT |
| ms2_library_path | path to the MS2 library (.msp or .pickle) | |
| ms2_sim_tol | 0.7 | MS2 similarity tolerance |
| fuzzy_search | yes | whether to perform fuzzy search |
| sample_normalization | no | whether to normalize the data based on total sample amount/concentration |
| signal_normalization | no | whether to run feature-wised normalization to correct systematic signal drift |
| run_statistics | no | whether to perform statistical analysis |
| plot_bpc | yes | whether to plot base peak chromatograms |
| plot_ms2 | yes | whether to plot mirror plots for MS2 matching |
| plot_normalization | yes | whether to plot normalization results |
| quant_method | peak_height | value for quantification and output, “peak_height”, “peak_area” or “top_average” |
More about parameters
Much more 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.