Proteome profiling of rat brain cortical changes during the early postnatal brain development using a surfactant-free protocol and label-free quantitation
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Introduction: High-resolution mass spectrometry and label-free quantitation (LFQ) was used in conjunction with a trifluoroacetic acid (TFA)-based, detergent-free sample preparation protocol to investigate brain protein changes in male and female rats during early postnatal rat cortical development. Protein ratios were obtained for the postnatal days (PND) 2, 8, 15 and 22, and the PND protein change profiles were constructed for key presynaptic, postsynaptic and adhesion cortical brain proteins. The profiles were compared to the analogous profiles assembled from published mouse and rat cortex fractionated-synaptosome data. The PND protein-change trendlines, Pearson Correlation Coefficient and linear regression analysis of the PND protein changes were used in the comparative analysis of the datasets. The analysis identified similarities and differences between four PND proteomic datasets.
Methods: Six individual male and female Long Evans pups for each PND were perfused with phosphate buffered saline and dissected. Rostral cortical tissues were stored frozen and digested with trypsin after dissolving them in TFA and performing cysteine-residues reduction-alkylation. Peptides were desalted using C18-SPE cartridges, their concentration was adjusted to 200 ng/μL and quantified using a LFQ nLC-MS data-dependent (MS/MS) method utilizing a high-resolution MS system, the 380–1,500 m/z full MS scan at the resolution of 120,000, and the MS/MS scan at the 60,000 (FWHM) resolution. Protein identification and LFQ was performed using the Proteome Discoverer-2.5TM software, with 37 processing software nodes, including the MSMS library, SEQUESTTM search and Precursor-Ion Quantifier nodesTM (two peptides per protein identification set minimum).
Preliminary data: Out of the 2,577 identified proteins, 1,917 male rat proteins and 1,894 female proteins produced at least one statistically significant (ANOVA adjusted p-value of 0.05 or less) non-zero LFQ log2 Pn/P22 ratio (n=2, 8, 15) over the studied rat PND experimental timeframe. The PND proteomic data comparison was performed between the current work data and the three previous studies: the nLC/MSMS mouse cortex synaptosomal fraction study (PNDs9, 15, 21) [Gonzalez-Lozano, et al, Sci. Rep. 2016, 6, 35456], the nLC/MSMS rat cortex synaptosomal and mitochondrial fractions study (PNDs1, 10, 20) [McClatchy, et al, J Proteome Res. 2012, 11, 2467], and the rat cortex proteomic study involving 2D-PAGE gel analysis (PNDs7, 90) [M. Wille, et al, J. Proteomics Bioinform. 2017, 10]. The synaptic and the mitochondrial protein PND ratios obtained from the current rat cortex work (PND2/PND22) correlated well with the PND ratios and the trendlines obtained from the previously quantified rat cortex synaptosomal cellular fractions (PND1/PND20): R² of 0.82 (male rat), 0.80 (female rat) and mitochondrial: R² of 0.81 (male rat), 0.83 (female rat). The synaptic protein PND ratios (current work, PND8/PND22) were correlated with the previous PND mouse cortex synaptosomal cellular fractions (PND9/PND21) ratios, showing R² of 0.71 (male rat vs mouse), and 0.72 (female rat vs mouse). This study showed that the early PND rat and the mouse synaptic protein profiles were similar, although mouse PND trendlines appeared flatter (the linear regression-correlation slope was 0.40). No definitive, significant rat male vs female protein ratio change trends were identified within the four investigated PND time points of PNDs2, 8, 15 or 22. The current study demonstrated that high-resolution LC-MS LFQ method in conjunction with the facile, TFA-based and detergent-free brain protein extraction technique can be confidently used for PND brain protein profiling (This abstract does not necessarily reflect USEPA policy).
Novel aspect: Early protein PND trends in male and female rat cortex by LFQ MS: current vs older cortical data comparison.