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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.

Periodic Reporting for period 1 - 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.)

Reporting period: 2019-11-01 to 2021-10-31

The aim of the project was to develop pull-down assay (PDA) methods initially working with a group in the Toxicology Centre at the University of Saskatchewan as the host organization and bring said methods to the Czech Republic. PDAs use nuclear receptor (NR) proteins that normally bind specific endogenous compounds but in exposure to xenobiotics that mimic endogenous compounds can potentially bind these compounds. The primary goal of the project was to develop a robust method to use these NRs to extract specific binding synthetic compounds from complex mixtures.
PDAs are an emerging field for environmental assessment and identification of endocrine disrupting compounds (EDCs). Simply put the pull-down assay is similar to a solid phase extraction however in a pull-down system the sorbent would be a NR. NRs represent one of the largest groups of transcription factors in vertebrates and compounds that interact with NRs, both natural and synthetic can cause an alteration in the metabolic function of an organism. EDCs represent a diverse group of chemicals of concern. These compounds do not represent one group in terms of use or structure, but all have the potential to mimic or disrupt the endocrine system. Because these compounds mimic existing low concentration natural hormones, they do not need to be in large concentrations to have an effect on the endocrine system. Concentrations in the body for some compounds at the low ng/kg level can be enough to cause non-normal outcomes in an organism. Today science is still working to understand the subtle effects of low-dose exposure from cocktails of EDCs. Exactly how many EDCs are in the environment is hard to tell as the number of compounds in use is ever growing while legacy and well described EDCs are still of concern.
The overall aim of the project was to develop a workflow for taking complex environmental samples and use PDAs and EDAs to reduce the complexity and focus just on those compounds that bind to a specific protein (Figure 1). The initial aim was to purchase pre-purified protein to be used in method development and in collaboration with the group in Saskatchewan and potentially the University of Toronto to synthesise additional material for use in larger scale tests. Further to this was the aim to trial additional purchased proteins using the same pull-down methodology adjusted to the unique features of the protein being used.
The project was initiated in November of 2019, and we investigated the existing equipment within the department for the feasibility of protein synthesis, bulk fractionation of samples for effect-directed analysis and the method development for pull-down assays. We considered an option to use either bound protein as in a pull-down assay or potentially a dialysis option developed by a post-doctoral student at the laboratory. Due to the covid pandemic starting in the Spring of 2020 it was decided to not purchase time-sensitive compounds like proteins and DNA for synthesis until we were sure of access to the laboratories. In March under the guidance of the Canadian/Saskatoon health authorities the Toxicology Centre was closed, and home office was the only option. During this period, the researcher learned computer programming on Python as a useful tool for assessment of large data sets as well as processed existing data on human exposure to synthetic compounds and published several manuscripts (a list of publications is included in the later text). The researcher also performed a literature search on some novel compounds of emerging interest and worked on predictive software to build a spectral database for high resolution mass spectrometry. He further developed previous collaborations with the University of Cartagena helping to supervise some PhD candidates and prepare related manuscripts.
In November 2020 the researcher was finally able to work again in the laboratories under strict safety conditions. Due to circumstances, it was decided not to synthase in-house proteins and to rely on commercial material for the project. To this end the focus of the work returned to the initial design of trialling a PDA for the NR pregnane X receptor (PXR).
Summary of deliverables:
• A pull-down assay for PXR was developed using known binding compounds to test the efficiency and identify whether differing buffer or elution methods would be more/less effective.
• All ethical requirements for use of human-derived proteins were maintained, and the researcher followed all the guidelines for the University host regarding safety and protocols established during the pandemic.
• All raw data files generated during this project have been securely duplicated and stored, and all results will be made publicly available.
• Once the PDA method has completed full and rigorous tests of which some have been achieved to date the process offers much for future exploitation both within the institutions the researcher worked at and with other groups.
The pull-down assays are innovative and valuable tools to add to the existing effect-directed and mass spectrometry tools. If these can be developed and made reliable, they will create a highly useful tool that other groups will wish to develop or be made available from either the host or home institution. The pull-down assay’s potential to bind and identify protein-specific endocrine-disrupting compounds is of global benefit. The method is being trialled for a wide range of matrices currently being explored with just EDA and traditional mass spectrometry methods in most instances but if the PDA is included in the workflow much more of the low-level, potentially unknown compounds may be identified.
An experience with the development of such versatile tools exploiting capacities of currently available ultra-high resolution mass spectrometers for more accurate investigation of EDCs will enhance future career perspectives of the researcher. It strengthened his interdisciplinary knowledge, programming and data processing skills as well as enhanced his collaborative network. Interactions with early-stage researchers at the hosting institution and beyond also brought useful experience exploitable in the future when the researcher will seek an academic position.
Currently, until the sensitivity of the method has been fully assessed in complex matrices it is difficult to say how robust the methodology is. In the future, pull-down assays may provide a key tool in analyses of endocrine disrupting chemicals in environmental and biological matrices but currently they are still until development.
There are multiple users of EDA and non-target methods who will find this versatile tool very useful. The researcher has been already engaged in discussions with scientists in China, Canada and Africa who all considered the pull-down assays potential enough to apply for their own grants building upon the ideas already envisaged.