Analytical and Bioanalytical Chemistry

, Volume 409, Issue 24, pp 5767–5778 | Cite as

Optimized experimental workflow for tandem mass spectrometry molecular networking in metabolomics

Research Paper


New omics sciences generate massive amounts of data, requiring to be sorted, curated, and statistically analyzed by dedicated software. Data-dependent acquisition mode including inclusion and exclusion rules for tandem mass spectrometry is routinely used to perform such analyses. While acquisition parameters are well described for proteomics, no general rule is currently available to generate reliable metabolomic data for molecular networking analysis on the Global Natural Product Social Molecular Networking platform (GNPS). Following on from an exploration of key parameters influencing the quality of molecular networks, universal optimal acquisition conditions for metabolomic studies are suggested in the present paper. The benefit of data pre-clustering before initiating large datasets for GNPS analyses is also demonstrated. Moreover, an efficient workflow dedicated to Agilent Technologies instruments is described, making the dereplication process easier by unambiguously distinguishing isobaric isomers eluted at different retention times, annotating the molecular networks with chemical formulas, and giving access to semi-quantitative data. This specific workflow foreshadows future developments of the GNPS platform.


Tandem mass spectrometry Metabolomics Molecular networking Natural products Annotation 



The authors are very grateful to E. Boyer, J. Smadja, S. Técher-Giraud, E. Girard-Valenciennes, I. Grondin, D. Strasberg (UMR C_53 PVBMT), and V. Dumontet for their contribution to plant material identification and species collection. We also would like to acknowledge G. Grelier for its helpful assistance for Python script redaction. This work has benefited from an “Investissement d’Avenir” grant managed by Agence Nationale de la Recherche (CEBA, ref. ANR-10-LABX-25-01).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

216_2017_523_MOESM1_ESM.pdf (5 mb)
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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Institut de Chimie des Substances Naturelles, CNRS UPR 2301Université Paris-Sud, Université Paris-SaclayGif-sur-YvetteFrance

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