Periodic Reporting for period 2 - 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.)
Okres sprawozdawczy: 2021-11-01 do 2022-10-31
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.
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.
At the beginning of the project we set objectives (i) to develop a standardized workflow for using an established pull-down assay with ultra-high resolution mass spectrometry and effect-directed analysis or other bioassay for streamlined detection of compounds of effect, (ii) to use additional nuclear receptors for use on the pull down assay methodology to determine if a single workflow is applicable, and (iii) to validate the method using environmental samples of varying complexity.
The project was initiated in November 2019, and we investigated the existing equipment within the hosting 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. The project implementation was complicated by the COVIDS-19 outbreak in March 2020 causing the long-term shutdown of the Toxicological centre. During the home office period, the researcher had a chance to improve his computer programming skills in Python as a useful tool for the assessment of large data sets. He also worked on processing existing data on human exposure to synthetic compounds and published several manuscripts. Namely, he further developed a previous collaboration with the University of Cartagena co-supervising some PhD candidates and contributing to related manuscripts. The researcher also performed a literature search on compounds of emerging interest and worked on predictive software to build a spectral database for high-resolution mass spectrometry. Starting November 2020, the researcher was able to work in the laboratories under strict safety conditions. Due to circumstances, it was decided not to synthesize 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). After returning to Czechia, the researcher continued to pursue the project objectives.
A pull-down assay was developed using known binding compounds, and various proteins were tested. They included RAR binding retinoic acids within the cell of almost all living organisms. In mammals it is linked to cell differentiation, immune function, haematopoiesis, reproduction, vision, and embryonic patterning. In plants it is a driver of root organogenesis. Though there are concerns regarding synthetic compounds and RAR interactions there is also investigations suggesting that algal blooms may also be sources of RAR interacting compounds. TTR as the thyroxine and retinol transporter was also used. It can be secreted in the liver and enter the blood stream or in the choroid plexus and enter the cerebrospinal fluid. As TTR transports thyroid hormones any disruption in children, for instance, can impact their brain development.
The project results and outputs include (i) 2 Gold Open Access publications and 3 manuscripts, (ii) presentations at four conferences, (iii) attendance of the researcher at the RSC-IITM Desktop Seminar on Environmental Sciences series and a lecture for students at RECETOX, (iv) co-supervision of PhD students.
The project was initiated in November 2019, and we investigated the existing equipment within the hosting 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. The project implementation was complicated by the COVIDS-19 outbreak in March 2020 causing the long-term shutdown of the Toxicological centre. During the home office period, the researcher had a chance to improve his computer programming skills in Python as a useful tool for the assessment of large data sets. He also worked on processing existing data on human exposure to synthetic compounds and published several manuscripts. Namely, he further developed a previous collaboration with the University of Cartagena co-supervising some PhD candidates and contributing to related manuscripts. The researcher also performed a literature search on compounds of emerging interest and worked on predictive software to build a spectral database for high-resolution mass spectrometry. Starting November 2020, the researcher was able to work in the laboratories under strict safety conditions. Due to circumstances, it was decided not to synthesize 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). After returning to Czechia, the researcher continued to pursue the project objectives.
A pull-down assay was developed using known binding compounds, and various proteins were tested. They included RAR binding retinoic acids within the cell of almost all living organisms. In mammals it is linked to cell differentiation, immune function, haematopoiesis, reproduction, vision, and embryonic patterning. In plants it is a driver of root organogenesis. Though there are concerns regarding synthetic compounds and RAR interactions there is also investigations suggesting that algal blooms may also be sources of RAR interacting compounds. TTR as the thyroxine and retinol transporter was also used. It can be secreted in the liver and enter the blood stream or in the choroid plexus and enter the cerebrospinal fluid. As TTR transports thyroid hormones any disruption in children, for instance, can impact their brain development.
The project results and outputs include (i) 2 Gold Open Access publications and 3 manuscripts, (ii) presentations at four conferences, (iii) attendance of the researcher at the RSC-IITM Desktop Seminar on Environmental Sciences series and a lecture for students at RECETOX, (iv) co-supervision of PhD students.
The PDA assays are innovative and valuable tools to add to the existing effect-directed and mass spectrometry tools. In the future, PDA may provide a key tool in analyses of endocrine-disrupting chemicals in environmental and biological matrices but currently, they are still in development. The researcher has been engaged in discussions with scientists from China, Canada, or Africa who all considered building upon the ideas already envisaged and developing PDAs further withing their own grants.
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 enhanced the career perspectives of the researcher. It strengthened his interdisciplinary knowledge, programming and data processing skills as well as 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.
The PDA methodology has proven to be a tool for analysis of unknown compounds in various matrices (water, food, dust) with a great potential. Once fully developed and validated, it opens exciting new research options as well as market opportunities. The PDA workflow, however, should be complemented by the ability to replicate proteins in a cost-effective manner. Currently, the further development is hindered by the lack of protein suppliers and high costs.
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 enhanced the career perspectives of the researcher. It strengthened his interdisciplinary knowledge, programming and data processing skills as well as 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.
The PDA methodology has proven to be a tool for analysis of unknown compounds in various matrices (water, food, dust) with a great potential. Once fully developed and validated, it opens exciting new research options as well as market opportunities. The PDA workflow, however, should be complemented by the ability to replicate proteins in a cost-effective manner. Currently, the further development is hindered by the lack of protein suppliers and high costs.