The processing wizard reduces the configuration of complex standardized workflows into few parameters. Those values are used to estimate or calculate all other parameters from spectral processing over feature detection and alignment to annotation and data export. The wizard organizes the different parts of the instrumentatl setup to define a workflow split up into: Sample introduction, IMS, MS instrument, workflow. More specific workflows are planned and we are open for ideas and contributions. Feel free to reach out if your workflow might be a candidate for a wizard setup.
The wizard is split into sections, most importantly, the MS part and the chromatography part. Parameters here reflect the sensitivity, resolution, and accuracy of these parts of the hyphenation. Selecting one of the default presets actually populates the initial values.
The defaults are only suggestions and different acquisition methods and instruments produce different noise levels etc. The raw data overview and aligned feature lists are useful to optimize these parameters.
Produces this batch:
Specify all data files that need to be processed
Sample introduction system¶
Depends on the selected sampling system, e.g., MALDI, HPLC, ...
Apply smoothing to the chromatograms
Stable ionization across samples¶
Used during feature grouping of adducts and other ions of the same molecule. Only use if matrix (e.g., salt) contents and ionization efficiencies are comparable across the whole study.
Crop retention time¶
Crops the chromatograms at these retention time values. Useful to cut off the start and the end of the chromatograms. The start is often diverted into the waist and the end often contains the cleanup procedure.
Max peaks in chromatogram¶
An estimate of the number of isomers and isobaric ions in the chromatograms. Used to estimate the percentage of data points that hold useful data for the feature resolving step. (Chromatographic threshold in local minimum resolver).
Minimum consecutive scans¶
Only keep chromatograms and features with at least X data points in retention time dimension.
Approximate feature FWHM¶
The full-width at half maximum of peaks in retention time dimension. Best extracted from the feature tables of already processed test datasets or from the raw data overview.
RT tolerance (intra-sample)¶
Retention time tolerance to group adducts and isotopes of the same molecule. The comparison is performed within each individual sample, usaully leading to small variance.
RT tolerance (sample-to-sample)¶
Retention time tolerance to align features across all samples. Dependent on retention time shifts.
Ion mobility instrument¶
Applies smoothing to the ion mobilograms
Minimum consecutive scans¶
The number of consecutive scans/datapoints in a valid ion mobilogram and feature
Approximate feature FWHM¶
The full-width at half maximum for IMS features
MS instrument, e.g., Orbitrap, qTOF, FTICR¶
Noise threshold (MS1/MS2)¶
Choose the mass detector from the drop down menu. Choose the Factor of lowest signal for centroided data where each spectrum contains noise signals, often represented by many signals at the same low intensity (spectral grass). This may correspond to static noise or single counts. Otherwise use an absolute intensity threshold.
Depending on the selected mass detector, separate absolute noise levels or factors are defined to threshold spectra of MS level 1 and 2 (or above). So the MS2 noise level is used for MSn data with level > 1. These parameters can be optimized by looking at the spectral raw data in the raw data overview.
Minimum feature height¶
The minimum height of chromatograms and features
Scan-to-scan m/z tolerance¶
Relative and absolute m/z tolerance. Always applies the maximum tolerance based on the m/z this means that smaller and higher values are stronger affected by the absolute and relative tolerance, respectively. Used to find the same signal in different scans when connecting chromatigrams. Reflects on the mass accuracy between scans in the same raw data file.
Feature-to-feature m/z tolerance¶
Relative and absolute m/z tolerance. Always applies the maximum tolerance based on the m/z this means that smaller and higher values are stronger affected by the absolute and relative tolerance, respectively. Used to group isotopes and adducts of the same molecule. Those m/z values are already averaged over their features and should have lower m/z differences that the scan to scan tolerance.
Sample-to-sample m/z tolerance¶
Relative and absolute m/z tolerance. Always applies the maximum tolerance based on the m/z this means that smaller and higher values are stronger affected by the absolute and relative tolerance, respectively. Used to align features across samples. Those m/z values are already averaged over their features but originate from differnt samples.
Original feature list¶
Options to either keep or remove the original feature lists. Keep is valuable during workflow optimization whereas remove saves resources and allows for more performant processing or large datasets. (See section on performance).
Min samples per aligned feature¶
Only keep aligned features that were detected in at least n samples. This parameter should usually scale with the sampleset size and if samples are relatively similar from their compounds. Uses the maximum of an absolute and relative value.
Only keep features with 13C¶
Detect isotope pattern and only keep feature with valid 13C isotope pattern.
Spectral library files¶
Select all spectral libraries to import and use during spectral library matching to annotate compounds in the final aligned feature list.
Local CSV database¶
- Specify the database file (csv or tsv format).
- Select m/z option to either
- calculate from given neutral mass (or formula/structure)
- use provided precursor m/z in column
- Filename column is only used for the library generation workflow
- Columns map the table column headers to the internal names in MZmine
Most of the parameters in the workflow section define data output and some workflow specific parameters.
- Define an export path and base file name, e.g., "D:\analysis\date_project" this will create a new folder and save all files from export modules there. Each module will add a specific suffix to the file name.
- Apply spectral networking (FBMN/IIMN): Will compare all MS2 spectra across features to form molecular networks by spectral similarity.
- Export for molecular networking (e.g., GNPS, FBMN, IIMN, MetGem): Will export all files for molecular networking
- Export for SIRIUS: Will export all files needed for SIRIUS
- Export annotation graphics: Exports annotations like spectral library matches, lipid matches, etc to graphical reports. Contains options to also export chromatographic/ion mobility shapes, images, and other plots.
Workflow parameters and descriptions¶
The data-dependent acquisition workflow is the default non target workflow. We recommend to also use this workflow for targeted analysis and combine it with the local CSV database search and spectral library search (Annotation).
More method and contributor metadata is required to build spectral libraries. This workflow produces reference libraries with options to filter and merge spectra.