An Integrated Workflow of Identification and Quantification on FDR Control-Based Untargeted Metabolome

Summary

We constructed an untargeted metabolomic workflow that integrated XY-Meta and metaX together. In this protocol, we displayed how to use XY-Meta to generate a decoy spectral library from open access spectra reference, and then performed FDR control and used the metaX to quantitate the metabolites after identifying the metabolomics spectra.

Abstract

Untargeted metabolomics techniques are being widely used in recent years. However, the rapidly increasing throughput and number of samples create an enormous amount of spectra, setting challenges for quality control of the mass spectrometry spectra. To reduce the false positives, false discovery rate (FDR) quality control is necessary. Recently, we developed a software for FDR control of untargeted metabolome identification that is based on a Target-Decoy strategy named XY-Meta. Here, we demonstrated a complete analysis pipeline that integrates XY-Meta and metaX together. This protocol shows how to use XY-meta to generate a decoy database from an existing reference database and perform FDR control using the Target-Decoy strategy for large-scale metabolome identification on an open-access dataset. The differential analysis and metabolites annotation were performed after running metaX for metabolites peaks detection and quantitation. In order to help more researchers, we also developed a user-friendly cloud-based analysis platform for these analyses, without the need for bioinformatics skills or any computer languages.

Introduction

Metabolites play important roles in biological processes. Metabolites are often regulators of various processes like energy transfer, hormone regulations, regulation of neurotransmitters, cellular communications, and protein post-translational modifications, etc^1,2,3,4. Untargeted metabolomics provides a global view of numerous metabolites^5,6. With advances in mass spectrometry and chromatography technologies, the throughput of metabolome MS/MS spectra is rapidly increasing in recent....

Protocol

1. Prepare metabolomics datasets for analysis

NOTE: In this demonstration, we use metabolomics datasets without QC sample. Data for case and control groups are needed. For demonstration, we use a public dataset in GNPS database²⁷.

1. Go to the webpage https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp. Click Browse Datasets.
2. Search the keyword "msv000084112" in the Title column. Click the dataset ID number for details and download the dataset using FTP.
3. Put the raw data in the folder /msv000084112.
   ​NOT....

Results

The raw data of msv000084112 was converted by msconvert.exe and generated mgf files (Supplementary Material S6).

XY-Meta generated GNPS-NIST14-MATCHES_Decoy.mgf file under /database folder. This is the decoy library generated from the original reference spectral library GNPS-NIST14-MATCHES.mgf. This decoy library can be reused. When reusing this decoy library, the user should set the decoy_pattern as 1 in parameter.default file, and set the decoyinput as the absolute path of t.......

Discussion

The FDR control of untargeted metabolites has been a great challenge. Here, we demonstrated a complete pipeline of large-scale untargeted metabolomics analysis (qualitative and quantitative) with FDR control. This effectively reduces the false positives, which are very common in MS analysis.

Preparing an appropriate reference spectral library for your study is a key point. A successful and sensitive MS/MS identification requires not only proper matching algorithms, but also proper reference sp.......

Materials

Name	Company	Catalog Number	Comments
GNPS	open source	n/a	https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp
XY-Meta	open source	n/a	https://github.com/DLI-ShenZhen/XY-Meta
metaX	open source	n/a	https://github.com/wenbostar/metaX
ProteoWizard	Free Download	3.0.22116.18c918b-x86_64	https://proteowizard.sourceforge.io/download.html
CHI.Client	Free Download	ndp48-x86-x64-allos-enu	http://www.chi-biotech.com/technology.html?ty=ypt