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.)
Période du rapport: 2019-11-01 au 2021-10-31
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.
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.
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.