Table of contents

Met4DX Documentation

Tutorial

Config Met4DX software

Met4DX Documentation

 

Introduction

Met4DX is a fast, robust, and user-friendly mass spectrometry data processing tool for metabolomics and lipidomics. The versatile tool facilitates the processing of both 3-dimensional LC-MS data and 4-dimensional LC-IM-MS data, encompassing main functions such as data conversion, peak detection, retention time correction, peak grouping, assignment of MS/MS spectra, and others.

Specifically, for peak detection, Met4DX employs a unique mass spectrum-oriented bottom-up assembly algorithm, ensuring the high-sensitive and high-coverage detection of TRUE MS peaks in metabolomics data (both 3D and 4D datasets). Additionally, Met4DX also supports a uniform peak quantification strategy that enhances the accuracy of peak quantitation in large-scale metabolomics analysis.

Moreover, Met4DX allows users to export results, enabling further analysis through complementary tools, such as MetDNA for metabolite identification, MetFlow for large-scale data integration, and MetTracer for stable-isotope tracing metabolomics data analysis.

Dependencies

Met4DX depends on the following software or SDKs to read the vendor data. Users who uses Met4DX are considered to agree with the licenses for the listed tools.

Recommended configuration

  • Processer: Core i7-12700 or above (4 core 8 thread and above are required)
  • Memory: 32 GB or above (at least 16 GB)
  • Resolution: 1080P (maximum 125% scaled, otherwise, some tools may not be displayed)
  • Operating system: 64-bit Windows 10 and above

Data format available for analysis

Vendor

Formats

Data reader

Agilent

MassHunter .d

ProteoWizard

Bruker

Compass .d, YEP, BAF, FID, TSF

ProteoWizard

TDF

TDF-SDK

Sciex

WIFF / WIFF2

ProteoWizard

Shimadzu

LCD (except ITOF)

ProteoWizard

Thermo

RAW

ProteoWizard

Waters

MassLynx .raw / UNIFI

ProteoWizard

 

 

Installation

Met4DX installer supports Windows 10 and above only currently. Please download the installer from the download page and install it directly. Please follow the installation wizard to install the software as you like.

Please contact us via metdna (AT) sioc.ac.cn if you have any problem.

NOTE:

  • Please make sure the software installation path and the windows account does not contains special characters, such as full-width characters, Chinese characters, etc.
  • Please confige Met4DX by setting correct 'Max processes' according your computer processer cores and free memory size (see Config Met4DX software for details)

 

 

Interface

Initial interface

Function bar

Project and data managements

  1. 1) Create a new project and add experiments for data processing.
  2. 2) Open a previously saved project or save the current project to a file.
  3. 3) Export results for downstream data analysis, e.g., MetDNA, MetFlow, and MetTracer, etc. when all added experiments are processed

Data processing tools

  1. 4) Load a parameter set with defined data analysis workflow or save parameters of current data analysis workflow to a parameter set.
  2. 5) Submit experiments for data processing using the current data analysis workflow or abort the processing for all submitted experiments.

experiments.

Other functions

  1. 6) Config Met4DX software.
  2. 7) View help documents.

 

Project setting

A project is required for data processing with Met4DX. Project means a bunch of experiments of different LC columns or ionization modes for certain samples acquired using the same MS instrument. Even if you have only one experiment, a project is still required. One should put all data together for the same project and submit the root directory to create the project and add corresponding experiments as follows:

Create a new project

Select the root director of the project root and name the project. If the project name is set as empty, a default project name will be set to the folder name of the project root.

Save current project

Save current project to a file (in .4dx format for Met4DX).

Open a previous project

Open a Met4DX project file (in .4dx format). Besides, Met4DX project file is registered to Met4DX software. Hence, one can double click the saved Met4DX project file to open it with Met4DX software.

 

Experiment setup

Add experiment

Experiments can be added with the ‘add experiment wizard’ by accepting ‘Add experiments now’ dialogue after creating the project or click the ‘Add experiment’ button from the function bar.

  • Set the name and directory of the experiment.
  • Setup the experiment by configuring LC column, MS type, Ion mode, etc. Please ensure the RT range and m/z range of MS1and MS2 acquisition are configured correctly for your own experiments.
  • Add files to the experiment including:
    • MS1 files
      • Sample files: [required] data files of subjected samples
      • QC files: [optional] data files of pooled quality control samples
      • Blank: [skipped] data files of blank samples, which will not be processed in current Met4DX
    • MS2 files: [optional] data files with MS2 data which may cause problems in peak alignment, such as segment DDA data
    • RT calibration: [optional] a csv table which records retention time of compounds in RTQC samples for RT calibration with RT in library during compound identification
    • Sample info: [skipped] a csv table of sample information with sample names, groups and injection order, which can be automatically generated in Met4DX 

NOTES:

  • Create a project before adding an experiment
  • When setting the file type on the right, you can select multiple files and right-click to set the file type in batches.
  • Files/folders without ‘set file types’ label (unsupported files/folders) will not be added to the experiment
  • Met4DX supports open MS data format (mzML and mzXML) and vendor raw data which can be converted to open MS data format with MSConvert. Data acquired with Bruker timsPro is also supported via timsTDF framework in Met4DX.
  • mzML is preferred if you have to provide open MS data format

​​​​​​Confirm sample information

The software will check the added files and group samples automatically according to the subfolders of MS1 files in submitted experiments after clicking ‘next’ button on the first page of ‘add experiment wizard’ and show the sample info in the second page. If there is no problem, one can click the ‘add’ button to add the configured experiment. The added experiments will be shown in project viewer in the top left panel of Met4DX.

If you have multiple experiments, please add them sequentially with the wizard.

NOTE:

  • If you have polarity switch experiments with Thermal instruments, such as Exploris 480, etc., two experiments with extended ion modes (positive and negative) will be created at the same time.

Parameter setting

Met4DX provides the most used parameter sets in Zhulab for different data processing tasks, which is highly recommended for inexperienced users, and even for all users. To start a data processing workflow, one should load a parameter set first, modifying parameters later, and submit experiment for data processing at last.

Load a parameter set

A parameter set contains multiple steps for data processing, which will be shown in the analysis workflow viewer after loading a parameter set. Parameters of each step can be configured by clicking corresponding steps. Key parameters will show by default and extended parameters will be shown and hidden by clicking the down and up triangle tool button in the button of the parameter configuration panel, respectively.

Save current parameter set

Users can save customized parameter sets according to actual needs for data processing next time with the data acquired with the same methods. Please configure the parameter set information using the pop-up dialogue window.

Parameter sets

NOTES:

  • Invalid parameter sets for your experiments will be ignored when loading parameter sets. If you found the desired parameter set can not be loaded, please check your experiment setup.
  • The DDA parameter set requires the data to be acquired using the DDA method, in which both MS1 and DDA MS2 are contained, and no additional MS2 files.
  • The segment DDA parameter set requires the data to contain extra MS2 data which is collected using segmented DDA method. In this case, segment DDA data (MS2 files) is submitted as MS2 file.

Parameter configuration

Parameters for each step are viewed and edited by clicking icon button of the corresponding step. Here is an example view:

NOTE：

  • The default parameters are highly recommended for all users, especially for inexperienced users.
  • Key parameters will be shown by default and extended parameters will be shown and hidden by clicking the down and up triangle tool button in the button of the parameter configuration panel, respectively.
  • All user added parameter sets can be edited in the software config window.

Data processing

Run analysis

  • Left click "Run Analysis": submit all experiments for data processing with selected analysis workflow.
  • Right click "Run Analysis": submit a single/all experiment(s) for data processing with selected analysis workflow.

After submission, the experiments in project viewer will show the corresponding processing status.

  •   current processing experiment.
  •   submitted experiment, waiting for processing.
  •   already processed experiment.

Processing plots

TIC plot (raw), RT shift plot, TIC plot (RT corrected) will be displayed during processing after corresponding steps finished.

These intermediate diagrams can be used to monitor data quality (such as RT shift, TIC consistency, etc.) and whether it has problems in data processing with submitted parameters.

Abort analysis

If you want to stop the analysis process, please click the 'Abort' button. In this case, the current processing task will be stopped and all other submitted but not finished tasks will be aborted.

 

Met4DX results

Default outputs

 The data processing results are stored in the results folder under the project folder for the corresponding experiment.

Export results

One can also export results for further analysis with other software tools from Zhulab, such as MetDNA, MetFlow, and even MetTracer for isotope tracing experiments. To export results, please click ‘Export Result’ button and select export type for your need.

NOTE:

  • MetTracer needs to export intermediate data, which is time consuming. Please be patient to wait for the export result finished notice.
  • This operation will export all experiment results at once. Hence, please make sure all added experiments are processed successfully before exporting results.

Tutorial

Demo datasets

  • Demo dataset 1: mouse liver tissues from Orbitrap Exploris 480
  • Demo dataset 2: fly head tissues from TripleTOF 6600
  • Demo dataset 3: NIST urine positive data for Bruker TIMSPro

Steps

  • Step1: Data preparation

Demo dataset 1: mouse liver tissues from Orbitrap Exploris 480

 

Demo dataset 2: fly head tissues from TripleTOF 6600

 

Demo dataset 3: NIST urine positive data for Bruker TIMSPro

  • Step2: Data importation

Demo dataset 1: mouse liver tissues from Orbitrap Exploris 480

 

Demo dataset 2: fly head tissues from TripleTOF 6600

 

Demo dataset 3: NIST urine positive data for Bruker TIMSPro

 

  • Step3: Parameter selection

Demo dataset 1: mouse liver tissues from Orbitrap Exploris 480

 

Demo dataset 2: fly head tissues from TripleTOF 6600

 

Demo dataset 3: NIST urine positive data for Bruker TIMSPro

 

  • Step4: Data processing

 

  • Step5: Result interpretation

Config Met4DX software

Software configuration

  • Max processes: Maximum number of threads during data processing. Please config it based on the cores and threads of the CPU and memory of the computer.
    • On average, one running process requires 1 GB and 2 GB memory for LC-MS and LC-IM-MS (Bruker timsPro instruments) data, respectively
    • Specifically, for Agilent and Waters instruments, much more memory will be required (~4-10 GB per process)
    • The max process should be configured with your free memory size and processer cores
  • Result folder name (CAUTION): The folder name of output results
  • Result temp folder: The folder name of temporary data. Please edit it when you do not have enough space in C drive.
  • Temp folder (CAUTION): Temporary folder path
  • Quant method (CAUTION): Method for calculating peak area
  • MSConvert (CAUTION): MSConvert path. One can use their own ProteoWizard for converting the raw data to temp mzML.
  • TimsData (CAUTION): TimsData library (TDF-SDK) path for reading Bruker TIMSPro data for LC-IM-MS data analysis.

Parameter set config

One can rename, delete, or modify information of the parameter sets saved by the user. Detailed value of parameters can also be configured in this configuration. Please save the parameters before you leave it. Otherwise, the modification will be ignored.

The help column of the parameter configuration view shows the meaning of each parameter. If you have problems, please contact us for clarification.

  • 021-68582292
  • 100 Haike Rd,Building 13,Zhangjiang Hi-Tech Park,Pudong New Area,Shanghai,China
Copyright © 2016 Zhu MS Lab, IRCBC. All rights reserved ​Technical Support: kuujiasoft