PullEd-MS · Finding unknown endocrine disrupting compounds through target pull-down assay filtration, effect direct analysis and ultra-high resolution mass spectrometry for a comprehensive efficient workflow.
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2019-11-01 → 2022-10-31
- EU contribution
- €242,522
- Participants
- 2
- Scheme
- MSCA-IF
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Results in brief
Finding unknown endocrine disrupting compounds through target pull-down assay filtration, effect direct analysis and ultra-high resolution mass spectrometry for a comprehensive efficient workflow.
The exposure of the living organism to xenobiotics mimicking endogenous compounds can potentially lead to binding these xenobiotics to nuclear receptors. The project focuses on the development of pull-down assays (PDA) using nuclear receptor proteins binding specific endogenous compounds for analysis of anthropogenic chemicals. This study is of interest for a society because we are in a situation where there are new chemicals being developed and released every day. Thousands of these have some potential uses for a society, and many may replace chemicals listed as potentially harmful. However, many of these compounds (and/or their degradation products) can also pose a significant environmental and health risks. Keeping up with monitoring emerging and legacy chemicals across all the environmental matrices is currently an impossible task. The aim of the project was to develop PDA methods for analysis of anthropogenic chemicals with potential biological effects. The two main screening tools being used today for monitoring of such substances are based on biological (assessment of bioactivity) or chemical (full scan data acquisition known also as non-target) screening. The bioactivity measurement process is a top-down approach relying on a biological response to a sample to focus the mass spectrometry searches to known compounds of effect. This approach often left a significant proportion of the bioactivity in a sample coming from unknown sources. The chemical approach is more of a catch-all process, but the outcome of such bottom-up approach is simply too much information. The PDA may provide the missing link between toxicological and non-target analysis. In this technique, a nuclear receptor protein or other cellular protein is synthesised and used as a bait to catch compounds of interest. Any compound that binds to the ligand-binding domain of a protein will then have an effect on the protein function.
Data: CORDIS, © European Union
Project objective
There are thousands of anthropogenic compounds in general use across the globe and every year more chemicals are created. For every compound, that finds its way into the environment multiple degradation compounds can result due to environmental processes. These changes alter the toxicological properties and chemical mobility creating potentially unpredicted effects. Environmental monitoring using effect direct analysis (EDA) to identify toxicological endpoints combined with mass spectrometry (MS) to detect the most abundant active compounds is an established methodology. The latest MS instruments the ultra-high resolution MS (UHRMS) have allowed full scan acquisition to become the cutting edge in the detection and identification of unknown compounds. However, knowing which EDA to apply is still a challenge and when UHRMS scans detect thousands of compounds identifying which compounds caused the toxicological response is a challenge. This MSC proposal aims to develop the use of pull-down assays linked to specific nuclear receptors (NR) such as estrogen receptor α as a filter to bind only those chemicals with an affinity for that specific protein. By then applying only the target specific EDA and fractionation to identify the most bioactive fraction before UHRMS a more focused workflow for compound detection based on effect can be developed. This workflow will be tested using samples of varying complexity including wastewater, sediment, and human biological samples. These workflows will then be tested using water collected from the Global Water Network to identify novel compounds of toxicological effect in the environment and using human urine from pregnant mothers collected from the CELSPAC-TNG cohort for identification of human exposure. In developing this comprehensive methodology, the sample will have specific toxicological effects identified as well as chemical investigation providing a thorough NR specific determination of both known and unknown compounds.
Original text from CORDIS.
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Data: CORDIS, © European Union