1. The document discusses non-target screening approaches for holistic chemical monitoring and discovery of unknown compounds using open science resources and real-time and retrospective mass spectrometry-based methods. 2. Key resources discussed include MassBank for sharing MS/MS spectra, the NORMAN suspect list exchange for sharing lists of compounds of interest, and the CompTox Chemistry Dashboard for accessing curated lists of chemicals. 3. Examples are given of applying these methods and resources to detect previously unknown chemicals in environmental samples and to retrospectively screen archived samples.

1. 1
Non-target Screening for Holistic Chemical Monitoring and
Compound Discovery:
Open Science, Real-time and Retrospective Approaches
Emma L. Schymanski
Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg.
Email: emma.schymanski@uni.lu
Reza Aalizadeh, Nikiforos Aligizakis, Juliane Hollender, Martin Krauss, Tobias Schulze,
Jaroslav Slobodnik, Nikolaos S. Thomaidis, Antony J. Williams
Image © www.seanoakley.com/
The views expressed in this presentation are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency.
https://tinyurl.com/rome-solutions-nts

2. 2
What chemicals are out there? What to monitor?
Numbers from Hollender, Schymanski, Singer & Ferguson, 2018, ES&T Feature, 51:20, 11505-11512. DOI: 10.1021/acs.est.7b02184
Chemical Space
Knowns, Suspects
and Unknowns
*Estimate from MINE predictions of
smaller datasets. Jeffryes et al.
2015. DOI: 10.1186/s13321-015-0087-1
**True number cannot be calculated.
Non-target
Screening of
Complex
Samples with
HR-MS

3. 3
Non-target Screening for Chemical Monitoring
Mod. from Hollender, Schymanski, Singer & Ferguson, 2018, ES&T Feature, 51:20, 11505-11512. DOI: 10.1021/acs.est.7b02184
Retrospective Retrospective
Real-Time
1) Open Science
2) Real-Time &
Retrospective
Screening
suspects Chemistry
Dashboard
Sampling AnalysisAnalysis
Data
Pre-Processing
Prioritization Identification

4. 4
1) Open Science: MassBank EU
https://github.com/MassBank/MassBank-data; https://github.com/MassBank/MassBank-web/; Rösch et al DOI 10.1021/acs.est.5b05186
http://massbank.eu/MassBank
o MassBank.EU was founded late 2012, hosted at UFZ, Leipzig, Germany
o >16,000 MS/MS spectra; 1,200 substances from NORMAN members
o MassBank now has >46,000 spectra from 32 contributing institutes!
o Thorough Github-based modernization in progress for traceability:
o Tentative/unknown/literature spectra (Level Scheme) as SI for publications
Schymanski et al DOI: 10.1021/es5002105

5. 5
NORMAN Suspect List Exchange
o http://www.norman-network.com/?q=node/236
o 21 lists available … specialist collections to market lists
• Integrated in NORMAN Databases & CompTox Chemistry Dashboard
Schymanski, Aalizadeh et al. in prep; https://www.researchgate.net/project/Supporting-Mass-Spectrometry-Through-Cheminformatics

6. 6
NORMAN SusDat
http://normandata.eu/normansusdat/
Schymanski, Aalizadeh et al. in prep.

7. 7
CompTox Chemistry Dashboard
https://comptox.epa.gov/dashboard/chemical_lists/ … new lists are released all the time!
http://normandata.eu/normansusdat/

8. 8
…All Combined in MetFrag
https://msbi.ipb-halle.de/MetFragBeta/ AND https://comptox.epa.gov/dashboard/dsstoxdb/batch_search (MetFrag Export)

9. 9
…All Combined in MetFrag
Example from: Schymanski & Williams, 2017, ES&T, 51 (10), pp 5357–5359. DOI: 10.1021/acs.est.7b01908

10. 10
Non-target Screening for Chemical Monitoring
o Part 2: Real-Time and Retrospective Screening
Retrospective Retrospective
And
Real-Time
Sampling Analysis
Data
Pre-Processing
Prioritization Identification
Mod. from Hollender, Schymanski, Singer & Ferguson, 2018, ES&T Feature, 51:20, 11505-11512. DOI: 10.1021/acs.est.7b02184

11. 11
Real-time Monitoring of the Rhine River
Hollender, Schymanski, Singer & Ferguson, 2018, ES&T Feature, 51:20, 11505-11512. DOI: 10.1021/acs.est.7b02184
Previously unknown chemicals detected due to “stand-out” patterns

12. 12
Real-time Monitoring of the Rhine River
Hollender, Schymanski, Singer & Ferguson, 2018, ES&T Feature, 51:20, 11505-11512. DOI: 10.1021/acs.est.7b02184
Previously unknown chemicals detected due to “stand-out” patterns

13. 13
European (World-)Wide Exchange of Suspects
Schymanski et al. 2015, ABC, DOI: 10.1007/s00216-015-8681-7; Wang et al 2016 Nature Biotechnology, DOI: 10.1038/nbt.3597
NORMAN Suspect List Exchange:
http://www.norman-network.com/?q=node/236
Tentatively Identified Spectra:
http://goo.gl/0t7jGp
Hits in GNPS MassIVE datasets:
TPs in skin: http://goo.gl/NmO4tx
Surfactants: http://goo.gl/7sY9Pf

14. 14
Retrospective Screening: Emerging Suspects
Alygizakis et al. 2018 ES&T, DOI: 10.1021/acs.est.8b00365 AND https://comptox.epa.gov/dashboard/chemical_lists/normanews
Switzerland
Mapimages©GoogleMaps
NormaNEWS in Science: DOI: 10.1126/science.360.6389.616-d

15. 15
Interactive heatmap available at http://norman-data.eu/NORMAN-REACH
NORMAN Digital Sample Freezing Platform
Retrospective screening of REACH chemicals in
Black Sea samples (various matrices)

16. 16
NORMAN Digital Sample Freezing Platform
“Live” retrospective screening of known and unknown
chemicals in European samples (various matrices)
http://norman-data.eu/ AND Aligizakis et al, in prep.

17. 17
NORMAN Digital Sample Freezing Platform
“Live” retrospective screening of known and unknown
chemicals in European samples (various matrices)
Future work: use results of unknowns to drive prioritization efforts
http://norman-data.eu/ AND Aligizakis et al, in prep.

18. 18
Acknowledgements I
Updates: https://www.researchgate.net/project/Supporting-Mass-Spectrometry-Through-Cheminformatics
emma.schymanski@uni.lu
Further Information:
http://www.norman-network.com/?q=node/236
https://massbank.eu/MassBank/
https://github.com/MassBank/
https://comptox.epa.gov/dashboard/
http://normandata.eu/normansusdat/
http://norman-data.eu/
.eu
EU Grant
603437
suspects Chemistry
Dashboard

19. 19

20. 20

21. 21
Creating High-Quality Mass Spectra
Stravs, Schymanski, Singer and Hollender, 2013, Journal of Mass Spectrometry, 48, 89–99. DOI: 10.1002/jms.3131
Automatic MS and MS/MS
Recalibration and Clean-up
Remove interfering peaks
Spectral Annotation with
- Experimental Details
- Compound Information
https://github.com/MassBank/RMassBank/
http://bioconductor.org/packages/RMassBank/
16,004 (61 %*) MS/MS spectra
1,269 (18 %*) substances
*% of all open LC-MS/MS data

22. 22
Confidence Levels for Tentative Structures
Schymanski, Jeon, Gulde, Fenner, Ruff, Singer & Hollender (2014) ES&T, 48 (4), 2097-2098. DOI: 10.1021/es5002105
o Annotation is the key to communicating information
MS, MS2, RT, Reference Std.
Level 1: Confirmed structure
by reference standard
Level 2: Probable structure
a) by library spectrum match
b) by diagnostic evidence
Identification confidence
N
N
N
NHNH
CH3
CH3
S
CH3
OH
MS, MS2, Library MS2
MS, MS2, Exp. data
Example Minimum data requirements
Level 4: Unequivocal molecular formula
Level 5: Exact mass of interest
C6H5N3O4
192.0757
MS isotope/adduct
MS
Level 3: Tentative candidate(s)
structure, substituent, class MS, MS2, Exp. data

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NORMAN Suspect List Exchange
o http://www.norman-network.com/?q=node/236
Schymanski, Aalizadeh et al. in prep; https://www.researchgate.net/project/Supporting-Mass-Spectrometry-Through-Cheminformatics
ReferencesFull Lists