The Metabolite Online Spectrum Analysis Center (METABOseek) is an interactive, browser-based tool to analyze and browse your mass spectrometry data. Here is a quick introduction to the basic functions.

1 Install METABOseek

1.1 System requirements

Recommended minimal system requirements:

  • Quad-core processor (or dual core with 4 threads)
  • 8 GB of RAM (16 GB or more are preferred)
  • Recent versions of Firefox or Chrome web browsers (METABOseek should work on any browser, but testing is done for these two)

We recommend computers with a monitor with at least full HD (1920 x 1080 pixels) resolution. You can use the zoom function of your web browser to scale the interface to your liking.

All files are loaded into memory, so that browsing will be very quick: It is easy to look at extracted ion chromatograms (EICs) for many MS features of interest across dozens of files within a fraction of a second. However, the initial loading of the data will take some time, and you may experience issues if you load many files at a time. We strongly recommend using centroided data files, as they will have a smaller memory footprint. Loading 50 data files from 20-minute high resolution LC/MS data acquisition should not be a problem on a computer with 16 GB of RAM.

1.2 Use the web version

If you have trouble installing METABOseek or want to just try it out with a restricted set of features (e.g. no xcms analysis), use the web version (allow some time (up to a minute) for the app to load completely).

1.3 Install on Windows

    Download the installer in the Download section and run it on your computer.

    Alternatively:

  1. Download the .zip file from the Download section.
  2. Unzip the file on your computer.
  • This may take a while!
  1. Run METABOseek by executing METABOseek.exe In the unzipped folder.
  2. This download might not give you the very latest version of METABOseek. You can update METABOseek to the newest version from inside the METABOseek app (Update/Help section in the navigation bar on the left).

1.4 Install on Mac / Linux

  1. Download and install R.
  2. Mac users: Get Xcode.

  3. Run R, enter this line:

    source("http://mosaic.bti.cornell.edu/files/install_METABOseek.R")
  • This should install the newest version of METABOseek. You can always update METABOseek to the newest version from inside the METABOseek app (Update/Help section in the navigation bar on the left).

  1. To run METABOseek, enter these lines into your R console:

    library(METABOseek)
runMseek()

1.5 Experienced R users (Windows, Mac or Linux):

If you have installed R (and the devtools package) already, you can install METABOseek like this:

devtools::install_github("mjhelf/METABOseek", ref="main")

If you want to make sure you get all the required packages, run the install script with this line:

source("http://mosaic.bti.cornell.edu/files/install_METABOseek.R")

2 Quickstart

A two-minute video demonstrating some data analysis with METABOseek.

3 Data analysis with METABOseek

With METABOseek, you can quickly visualize data from batches of high-resolution LC/MS data files and find differences between groups of samples. It is not necessary to do any analysis before looking at your data, but a typical workflow starts with a data analysis step:

  • Detection of MS features (defined by m/z and retention time) with the xcms analysis module
  • alternatively, you can use an MS feature list that you generated elsewhere.
  • Molecular networking of MS2 spectra on the GNPS website to be used in the METABOseek MS2 browser.

Then, use METABOseek to browse the data, find molecular features of interest, predict the molecular formula and make structure predictions based on MS2 data.

3.1 Overview

METABOseek is structured into two major sections, the Data Explorer section for visualization and statistical analysis tasks, and the XCMS analysis to identify LC/MS features in MS data files. You can switch between these sections with the navigation menu on the left of the screen. Inside each section, contents are arranged in boxes starts up on the data explorer page, so that you can quickly load MS data upon startup. To get to the XCMS

3.2 Data Explorer

At the heart of METABOseek is the interaction between data visualization in the “Data viewer” box, and a table of LC/MS data features in the Feature table box

3.2.1 Load MS Data into METABOseek

In most cases, the first thing you want to load is some LC/MS data to look at. You can load the files from the Grouped EICs tab in the Data viewer box:

The METABOseek start page

The METABOseek start page

1. Select a folder with MS data files.

All files with supported file extensions in the selected folders and all its subfolders will be imported, so it makes sense to pre-sort your files in a reasonable folder structure. Note that here, unlike for the xcms analysis, you may want to include widely different samples (e.g. blanks, to visually confirm they do not contain a compound of interest).

2. Load a METABOseek project folder.

When you run xcms through METABOseek, the program generates a project folder that contains the results from that xcms analysis run, and all settings that were used in it. If you select a project folder, all MS data files from an analysis will be loaded and grouped in the same way you set up for the xcms analysis. METABOseek will then ask you which feature table you want to load from the project folder, and will load it in the Feature table box.

3. Load a .zip file with MS data files.

It will be unzipped to a temporary folder and all supported files in it will be loaded into METABOseek.

Data files with MS/MS data can be loaded along with files not containing MS/MS data. Use the MS2 browser tab in the Data viewer box to find and compare MS2 scans related to features in the Feature table, or load a GNPS MS/MS networking result file to look at related MS2 spectra.

3.2.2 Load feature tables

3.2.3 Load a GNPS MS2 data network

If you have analyzed your MS/MS data on the GNPS website, you can load the result .zip file into METABOseek directly:

Go to the MS2 browser tab in the Data viewer box, check the “Show network” checkbox and use the Load GNPS network (.zip) menu to browse for a .zip file on your computer. Alternatively, you can use the Load node table and Load edge table menus to load a network (with the Load Network button below).

3.2.4 Visualize EICs

3.2.5 Interactive MS data view

3.2.6 View MS/MS data

If you have loaded MS data files which contain MS/MS data, go to the MS2 browser tab in the Data viewer box to look at MS/MS scans in one of two ways:

1. Use the feature table

When you select one (or multiple) entries in the Feature Table, METABOseek will find any MS/MS scans that have a parent mass matching the selected Feature Table entry (i.e. currently within 5 ppm and 200 seconds, soon to be customizable).

2. Use a GNPS network

If you have loaded a GNPS network file, you can also display spectra for each network node, as long as the files used to generate the network are loaded into METABOseek. Navigate from the network overview to a contingent network cluster (or “subgraph”) by clicking on it while holding the SHIFT key. The view will now zoom in on the subgraph. You can use the control menu above the network to show node and edge labels of your choice (e.g. Parent m/z and m/z difference between nodes (“deltaMZ”)), and apply a coloring scheme (e.g. color by default groups). To select a node and display all MS/MS spectra associated with it, click on a node while holding the SHIFT key.

You can move nodes by dragging them with your mouse while holding the CTRL key (this helps make all labels visible in a dense network). Return back to the network overview by clicking on the graph while holding the Z key.

Manage and compare MS2 spectra

All MS/MS scans matching a selection (from a network or from the Feature Table) are shown in a table in the MS2 browser tab. An average spectrum of all scans shown in this scan table is displayed on the left. You can select single or multiple scans in the scan table to show the spectrum of only the selected scan(s).

MS/MS spectra are shown on the right side. You can choose to keep a spectrum view - it will then not be overridden when you select a new Feature table entry or network node. You can show up to 5 spectrum views at the same time. By default, all peaks that occur in more than one of the shown spectra are highlighted in blue. You can disable this comparison with the Compare checkbox. You can also download the shown spectrum views in .pdf format by clicking Download spectra.

3.3 XCMS analysis

This section will help you to set up an xcms analysis in METABOseek in order to identify LC/MS features that are differential between sets of data files. This can, for instance, be useful to assess the impact of a mutation on the metabolome of an organism or to identify compounds associated with the activity of an enzyme.

Running an xcms analysis - a description of the highlighted steps is below.

Running an xcms analysis - a description of the highlighted steps is below.

  1. Select a folder with MS data files. All files with supported file extensions in the selected folders and all its subfolders will be listed, so it makes sense to pre-sort your files in a reasonable folder structure:
  • All files should be acquired under comparable conditions, especially with the same polarity. Differences in LC gradient or general composition (e.g. through widely different extraction methods, or comparing samples and blanks) can also make it difficult to find differential features.
  1. There are 7 tables with xcms settings you can change here. Navigate through them with the drop down menu highlighted as (2.). A short description for each parameter is given when you hover over the table entries. You can use the default settings and proceed to step 3 without changing any of them. The default is for highly similar LC/MS runs acquired at high resolution and high accuracy (< 5 ppm), and will find relatively small peaks (even if they only occur in a single replicate) with a relatively long runtime.
  • Output files: select which output files you want to get. Output files settings
  1. Start the analysis
  2. You can save settings as a .zip file (on windows computers, 7-zip or other software allowing for the zip command line prompt must be installed), or load a .zip file with settings from a previous run.
  • Note that this will override your selection of MS data files. If you want to apply the settings to a new set of data files, load the settings first and then select a folder (step 1).

3.4 Supported file types

METABOseek uses the MSnbase and xcms packages to load files of the following formats:

  • .cdf
  • .nc
  • .mzData
  • .mzML
  • .mzXML