Experimental procedures
Mouse proteomics assays are described in detail in Marques et al.(2022d). Briefly, a group of healthy male C57BL/6J mice was treated with
1 mg kg-1 MTK by oral gavage, once a day, for a week.
Brain, hippocampus, and prefrontal cortex were collected and used for
protein extraction. Samples were analysed by ultra-performance liquid
chromatography coupled to high resolution mass spectrometry with
electrospray ionization UPLC‑HRMS-ESI and the raw data were deposited in
public data repositories (Marques et al. , 2022b). The
well-characterized C57BL/6J mouse strain is the strain more commonly
used for safety and toxicity assessment, and is also refractory to the
development of many tumours, making it an ideal strain for metabolic and
omics studies on non-tumorigenic toxic effects (Kalueff and Nguyen,
2014; Mouse Genome Sequencing Consortium, 2002; Sarsani et al. ,
2019).
A detailed description of neuron proteomic assays is also available in
Marques et al. (2022d). A chicken neuron-enriched cell model was
exposed to MTK and cells were collected for protein extraction. Samples
were analysed by UPLC‑HRMS-ESI and raw data deposited in public data
repositories (Marques et al. , 2022a). Brain-expressed chicken
amyloid-β has a higher homology to the human form than that of mouse.
Since amyloid-β is one the proteins involved in Alzheimer’s disease, for
which the repurposing of MTK is being considered, we developed an
optimised chicken-based neuron model (Carrodeguas et al. , 2005)
for assessment of MTK‑driven proteomics alterations.
Data processing
Raw data were re-processed according to the parameters presented in
Marques et al. (2022d). Mouse and chicken proteomics data were
analysed on MaxQuant software v2.0.3.0 (Cox and Mann, 2008; Tyanovaet al. , 2016a) using the internal search engine Andromeda (Coxet al. , 2011), and Uniprot (UniProt Consortium, 2020) databases
restricted to specific groups of proteins from Mus musculus(Proteome UP000000589; keywords “ADRs (anxiety + stress + sleep +
depression + suicide) + apoptosis + inflammation + leukotrienes +
metabolism + microglia + neurons + repurposing (amyloid, synuclein, tau,
APP, BACE, Psen1, Psen2, nicastrin, neprilysin, adam 17)” + tissue
name ”) or Gallus gallus (Proteome UP000000539, keywords
“ADRs (anxiety + stress + sleep) + inflammation + leukotrienes +
neurons + repurposing (amyloid, synuclein, tau, APP, BACE, Psen1, Psen2,
nicastrin, neprilysin, adam 17 )”). MaxQuant output was processed using
Perseus v1.6.15.0, with default settings (Tyanova et al. , 2016b).
Significantly altered proteins (|fold change| ≥ 1.5
and p < 0.05) were annotated using PANTHER v16.0 andMus musculus or Gallus gallus reference lists (Mi et
al. , 2021).