The SIMPLIFY Protocol: A Monophasic Extraction System Suitable for Exposomics, Metabolomics, Lipidomics, and Proteomics Research

Anh Hoang Nguyen; Victor Castro-Alves; Emilia Holmström Flores; João Marcos G. Barbosa; Matilda Kråkström; Päivikki Reinivuori; Otto Kauko; Alex Dickens; Matej Orešič; Tuulia Hyötyläinen

doi:10.1021/acs.analchem.5c05322

The SIMPLIFY Protocol: A Monophasic Extraction System Suitable for Exposomics, Metabolomics, Lipidomics, and Proteomics Research

Anh Hoang Nguyen
, Victor Castro-Alves^*
, Emilia Holmström Flores
, João Marcos G. Barbosa
, Matilda Kråkström
, Päivikki Reinivuori
, Otto Kauko
, Alex Dickens
, Matej Orešič
, Tuulia Hyötyläinen^*

^*Corresponding author for this work

Turku Bioscience Centre

Research output: Contribution to journal › Article › Scientific › peer-review

Abstract

Advancing our understanding of human health and disease requires comprehensive analytical approaches capable of capturing the complex interplay between endogenous metabolism and environmental exposures. A major challenge in clinical research is the ability to capture multidimensional data, particularly a broad range of biochemical profiles, due to limitations of biological resources, time, and budget. In this study, we introduce the SIMPLIFY Protocol, a unified monophasic extraction method that enables the simultaneous extraction of chemical exogenous products and endogenous molecules. The method was evaluated against in-house extraction techniques, including protein precipitation with methanol (MeOH) and acetonitrile (ACN), and the Folch method using various sample types, particularly certified reference materials. We demonstrate that the SIMPLIFY Protocol not only performs comparably to our in-house methods but also offers enhanced versatility for additional applications such as derivatization and proteomics. The analyte abundances and reproducibility with this method strongly correlate with those from in-house-established techniques across diverse sample types. The method encompasses a broad spectrum of compounds, effectively profiling approximately 800 identified compounds, including polar compounds (e.g., amino acids), semipolar compounds (e.g., polyfluorinated compounds, bile acids, and lysophophatidylcholine), and nonpolar compounds (e.g., cholesteryl ester), with some limitations in extracting triacylglycerols. By maintaining simplified workflow and minimizing biological and resource consumption of multiple extractions, this method supports high-throughput exposomics/metabolomics and lipidomics studies. Furthermore, its streamlined design facilitates (semi)automation, making it highly suitable for large-scale clinical studies, where efficiency, cost-effectiveness, and sample availability are critical factors.

Original language	English
Pages (from-to)	26175-26183
Number of pages	9
Journal	Analytical Chemistry
Volume	97
Issue number	47
DOIs	https://doi.org/10.1021/acs.analchem.5c05322
Publication status	Published - 2 Dec 2025
MoE publication type	A1 Journal article-refereed

Funding

The authors thank Lucas Landmann, Hannah Freyer, and Melissa Lausberg for their support in sample extraction at the optimization step. The study received the support from the Swedish Research Council (grant nos. 2020-03674 and 2016-05176 to T.H. and M.O.), Formas (grant no. 2019-00869 to T.H. and M.O.), Novo Nordisk Foundation (grant no. NNF21OC0070309 to T.H.), by the “Investigation of endocrine-disrupting chemicals as contributors to progression of metabolic dysfunction-associated steatotic liver disease” (EDC-MASLD) consortium funded by the Horizon Europe Program of the European Union under Grant Agreement 101136259 (to T.H. and M.O.), and by the Swedish Knowledge Foundation (grant no. 20220122, to T.H. and M.O.). The authors would also like to thank the Turku Metabolomics Centre and Biocentre Finland for funding the instruments at the University of Turku used in this manuscript. Views and opinions expressed are those of the authors only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them.

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the-simplify-protocol-a-monophasic-extraction-system-suitable-for-exposomics-metabolomics-lipidomics-and-proteomicsFinal published version, 2.45 MBLicence: CC BY

https://urn.fi/URN:NBN:fi-fe202601279146

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Nguyen, A. H., Castro-Alves, V., Holmström Flores, E., Barbosa, J. M. G., Kråkström, M., Reinivuori, P., Kauko, O., Dickens, A., Orešič, M., & Hyötyläinen, T. (2025). The SIMPLIFY Protocol: A Monophasic Extraction System Suitable for Exposomics, Metabolomics, Lipidomics, and Proteomics Research. Analytical Chemistry, 97(47), 26175-26183. https://doi.org/10.1021/acs.analchem.5c05322

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abstract = "Advancing our understanding of human health and disease requires comprehensive analytical approaches capable of capturing the complex interplay between endogenous metabolism and environmental exposures. A major challenge in clinical research is the ability to capture multidimensional data, particularly a broad range of biochemical profiles, due to limitations of biological resources, time, and budget. In this study, we introduce the SIMPLIFY Protocol, a unified monophasic extraction method that enables the simultaneous extraction of chemical exogenous products and endogenous molecules. The method was evaluated against in-house extraction techniques, including protein precipitation with methanol (MeOH) and acetonitrile (ACN), and the Folch method using various sample types, particularly certified reference materials. We demonstrate that the SIMPLIFY Protocol not only performs comparably to our in-house methods but also offers enhanced versatility for additional applications such as derivatization and proteomics. The analyte abundances and reproducibility with this method strongly correlate with those from in-house-established techniques across diverse sample types. The method encompasses a broad spectrum of compounds, effectively profiling approximately 800 identified compounds, including polar compounds (e.g., amino acids), semipolar compounds (e.g., polyfluorinated compounds, bile acids, and lysophophatidylcholine), and nonpolar compounds (e.g., cholesteryl ester), with some limitations in extracting triacylglycerols. By maintaining simplified workflow and minimizing biological and resource consumption of multiple extractions, this method supports high-throughput exposomics/metabolomics and lipidomics studies. Furthermore, its streamlined design facilitates (semi)automation, making it highly suitable for large-scale clinical studies, where efficiency, cost-effectiveness, and sample availability are critical factors.",

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doi = "10.1021/acs.analchem.5c05322",

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Nguyen, AH, Castro-Alves, V, Holmström Flores, E, Barbosa, JMG, Kråkström, M, Reinivuori, P, Kauko, O, Dickens, A, Orešič, M & Hyötyläinen, T 2025, 'The SIMPLIFY Protocol: A Monophasic Extraction System Suitable for Exposomics, Metabolomics, Lipidomics, and Proteomics Research', Analytical Chemistry, vol. 97, no. 47, pp. 26175-26183. https://doi.org/10.1021/acs.analchem.5c05322

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T2 - A Monophasic Extraction System Suitable for Exposomics, Metabolomics, Lipidomics, and Proteomics Research

AU - Nguyen, Anh Hoang

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AU - Holmström Flores, Emilia

AU - Barbosa, João Marcos G.

AU - Kråkström, Matilda

AU - Reinivuori, Päivikki

AU - Kauko, Otto

AU - Dickens, Alex

AU - Orešič, Matej

AU - Hyötyläinen, Tuulia

PY - 2025/12/2

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N2 - Advancing our understanding of human health and disease requires comprehensive analytical approaches capable of capturing the complex interplay between endogenous metabolism and environmental exposures. A major challenge in clinical research is the ability to capture multidimensional data, particularly a broad range of biochemical profiles, due to limitations of biological resources, time, and budget. In this study, we introduce the SIMPLIFY Protocol, a unified monophasic extraction method that enables the simultaneous extraction of chemical exogenous products and endogenous molecules. The method was evaluated against in-house extraction techniques, including protein precipitation with methanol (MeOH) and acetonitrile (ACN), and the Folch method using various sample types, particularly certified reference materials. We demonstrate that the SIMPLIFY Protocol not only performs comparably to our in-house methods but also offers enhanced versatility for additional applications such as derivatization and proteomics. The analyte abundances and reproducibility with this method strongly correlate with those from in-house-established techniques across diverse sample types. The method encompasses a broad spectrum of compounds, effectively profiling approximately 800 identified compounds, including polar compounds (e.g., amino acids), semipolar compounds (e.g., polyfluorinated compounds, bile acids, and lysophophatidylcholine), and nonpolar compounds (e.g., cholesteryl ester), with some limitations in extracting triacylglycerols. By maintaining simplified workflow and minimizing biological and resource consumption of multiple extractions, this method supports high-throughput exposomics/metabolomics and lipidomics studies. Furthermore, its streamlined design facilitates (semi)automation, making it highly suitable for large-scale clinical studies, where efficiency, cost-effectiveness, and sample availability are critical factors.

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KW - Peptides and proteins

KW - Metabolism

KW - Lipids

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JF - Analytical Chemistry

IS - 47

ER -

The SIMPLIFY Protocol: A Monophasic Extraction System Suitable for Exposomics, Metabolomics, Lipidomics, and Proteomics Research

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