Proteome profiling of rat brain cerebellum during the early postnatal brain development
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Background and Purpose: During the sensitive perinatal period, environmental stressors are a threat to brain development. We applied proteomics to investigate the protein changes occurring during the early postnatal rat cerebellum development.
Methods: High-resolution mass spectrometry and label-free quantitation (LFQ) was used to investigate brain protein changes in male and female rats. Six animals were in each of the Long Evans pup groups: male cage control (M0), male treated (M1), female cage control (F0), female treated (F1). The dams’ treatment was a mild 3.78 mg/kg of emamectin treatment given starting at GD6 through PND21.
Results: Out of the 2,522 proteins identified with high confidence in the 96 individual rat samples, 1,815 male rat proteins and 1,818 female proteins showed at least one statistically significant (ANOVA adjusted p-value ≤ 0.05) non-zero LFQ log2 P[n]/P22 ratio (n = PND 2, 8, 15). For these significant logarithmic ratios, the male vs the female and the treated vs untreated data comparisons showed the same high Pearson correlation coefficients (PCC) of 0.98 indicating that these data subsets were almost identical, hence these ratios were grouped together. Out of the 2,522 quantified proteins (40,384 abundance data points), 72% had CV % ≤ 20, while 19% had CV % between 20 and 40, indicating good data quality. There were both similarities and differences in these PND protein change patterns in rat cerebellum and the synaptosome-enriched fraction [1] (DB McClatchy et al, J. Proteome Res. 2012, 11(4), 2467). 98% of the protein PND ratio P2-P22 lines characterized by absolute value of the log2 Pn/Px ratios of >= 0.5 constructed using the n = 2, x = 22 (current work), and n = 1, x = 20 [1] showed the same log ratio signs, thus directional trends. 79 proteins displayed a “dip” and 251 a “bump”, at the P8/P22 ratio compared to the others. There were 560 PND trendlines possessing overlapping standard deviation brackets at P2/P22, P8/P22 and P15/P22, while 238 lines were consistently rising and 139 consistently falling. In comparison, of 149 trendlines reconstructed from [1], for the proteins that were in common with the current study, 29 demonstrated either a small “dip” or a flat area between the P1 and P10 points. However, these features did not correlate well with the ones of the current unfractionated cerebellum work. This might be due to the complex protein expression patterns in cerebellum, evident in the synaptosomal and mitochondrial fraction analysis [1]. The current data were compared to the previous rat cortex proteomic study involving the same animals (Winnik, et al, J. Proteome Res. 2023, 22(7) 2460), and the PCC were: 0.80-0.84 (P2/P22), 0.86 (P8/P22), and 0.87-0.90 (P15/P22), pointing to the weakest correlation for the P2/P22. This result can be explained by the larger magnitude of the early PND protein ratio change and by the developmental differences between rat cerebellum and the rostral cortex.
Conclusions: This study demonstrates effectiveness of the high mass resolution MS LFQ in postnatal day (PND) protein profiling using brain cerebellum samples. It sets the stage for the investigation of treated vs control experiments investigating toxico-proteomic environmental exposure endpoints in young rats, and for further hypothesis-based focused follow up studies. This abstract does not necessarily reflect US EPA policy.