FUNEL

The FUNEL module includes the following functions: (1) feature detection, (2) isotopes and adducts/fragments annotation, (3) feature alignment across samples, (4) filtering features using controls, (5) calculating exact mass based on adducts/fragments for compound database search.

1. Please log in and upload files before use.

   Data preparation is described in the MSConvert Usage section.

2. Check the parameter settings.

   The default parameters settings are designed to be compatible with Q-TOF systems from different manufacturers, such as Waters, SCIEX, Thermo, etc. Users may modify these parameters as needed for their particular analytical purposes.

   Parameter & Description	Default Setting
   Isotope match	False
   Signal intensity threshold for controls	1000
   Signal intensity threshold for samples	10000
   ppm tolerance	20
   Delta MZ	0.01
   Min peak width	9
   Max peak width	20
   Rt Check: delta retention time (in seconds) that is allowed to match two features within a sample	30
   Noise level for controls	100
   Noise level for samples	1000
   Calculate adducts/fragments	Ture
   SNR threshold: Features with signal-to-noise ratios lower than this value are not included in analysis	2
   perfwhm: This parameter is used to group two features depending on their retention time. Typically between 0.6 and 1.2. Default is 1.	1
   Remove control signals with stringent criteria: If true, rigorous filtering will be performed to ensure features that can be found in control groups are removed from samples of interest. This slows down the computation and may remove some true hits.	Ture
   RT tolerance: Delta retention time (in seconds) that is allowed to match two features between samples. Determined mainly by experimental conditions.	60
   Fold change filter: When the signal intensity in the sample of interest exceeds the signal intensity in the controls by a certain fold, such a signal is retained for analysis. If set to 0, signals detected in controls are all removed from samples of interest.	0
   Intensity threshold for adducts calculation	5000
   Context-based adducts/fragments calculation	Ture
   Polarity	Positive

3. Upload the corresponding files under the option.

   The Compound Library to Search option offers NPA and COCONUT databases. Users can choose either as needed; selecting "None" will search both databases.

   Data files for control: Choose your single or multiple control data files, support .mzXML format.

   Data files for sample of interest: Choose your single or multiple data files for samples of interest, support .mzXML format.

4. Enter a task name to start your analysis journey.

   Click "Tasks" at any time to check your analysis progress.

5. Data output.

   The output includes eight files. The .json file records all parameter settings from the user's submitted task, while FilteredList.csv provides the final results.

6. Data analysis. A brief explanation of the key output results in the FilteredList.csv file is provided below.

   Columns	Description
   mz, mzmin, mzmax	The m/z value of the metabolite and its fluctuation range within the file
   rt, rtmin, rtmax	The retention time (in seconds) of the metabolite and its fluctuation range within the file
   adducts, exact mass	Calculated adducts and exact mass of the metabolite
   BALF-D2-PBS-1, BALF-D2-PBS-2(An example of the file name)	The uploaded file name. Numbers in each column indicate relative abundances calculated from the peak intensities of the corresponding m/z values; “-1“ means the abundance falls below the defined threshold.
   match	The compound database search result for the corresponding metabolite m/z is output as an InChIKey (copyable to PubChem https://pubchem.ncbi.nlm.nih.gov/ for structure lookup); “-1” means no matching m/z found in the specified database.