Proteomics

Frozen homogenized whole brains obtained from newborn B × C maternally exposed female pups were submitted to the Molecular Education, Technology, and Research Innovation Center (METRIC) at NCSU for proteomic analysis. The brain samples used in the proteomic analysis originated from the four 0 ppm and four 50 ppm females represented in the RNA-seq analysis; additionally, four 1 ppm females representing four different litters were used for proteomic analysis.

Samples were quantitated for protein content using a Pierce bicinchoninic acid (BCA) kit and normalized to 50 μg of protein by diluting the appropriate amount of sample in 50 mM ammonium bicarbonate with 1% (w/w) sodium deoxycholate. Normalized samples were reduced with dithiothreitol (DTT) and alkylated with iodoacetamide (IAM) to break disulfide bonds and prevent reformation. Following this, a filter aided sample preparation (FASP) protocol^91,92 was used to purify the samples which were then treated with trypsin at a 50:1 protein:trypsin concentration. Samples were incubated for 4 h and then aliquoted for LC–MS analysis.

Chromatographic separation was achieved using a Thermo Scientific EASY nLC 1200 system (Bremen, Germany). Pico-frit columns were purchased from New Objective (Woburn, MA) and bomb packed to a length of 20–30 cm with reverse phase ReproSil-Pur 120 C-18- AQ 3 µm particles (Dr. Maisch, Germany). Two microliters of sample was injected and subsequently separated using a gradient of mobile phase A (98% water, 2% acetonitrile, and 0.1% formic acid) and mobile phase B (80% acetonitrile, 20% water 0.1% formic acid). The LC method consisted of a 120 min gradient with a linear ramp from 0% B to 40% B, a 1 min ramp to 100% B which was held for 6 min (123–129 min), followed by equilibration of the column at 0% B (130–140). A flow rate of 300 nL/min was used.

Orbitrap tandem mass spectrometry was performed using a Thermo Scientific Q-Exactive HF (Bremen, Germany) in a top 20 data dependent acquisition (DDA) mode, where the 20 most abundant precursors are selected for fragmentation per full scan. MS1 and MS2 scans were performed at a resolving power of 120,000 and 15,000 at m/z 200, respectively. A dynamic exclusion window of 20 s was used to avoid repeated interrogation of abundant species. Automatic gain control (AGC) was 1e6 and 1e5 for MS1 and MS2 scans, respectively. Samples were run in random order, and a quality control BSA digest was run every fifth injection to ensure proper LC–MS/MS reproducibility.

Resulting raw data were loaded into Proteome Discoverer (version 2.0). Spectrum files were run through the spectrum selector node to appropriately identify peaks and this data was collated using the Sequest HT node and aligned with a FASTA file which contained all proteins indexed by Uniprot and assigned a taxonomy ID of 10,090 (mus musculus). Oxidation (dynamic) and carbamidomethylation (static) modifications were considered in Sequest HT with a maximum of 2 potential missed cleavage sites and peptide lengths between 6 and 144 amino acids. The Percolator node was used for false discovery rate (FDR) calculations.

Data was then filtered to remove the following: abundance ratio adj. p-value (50 ppm vs 0 ppm) > 0.05, # unique peptides > 1, Abundances (grouped) CV% 50 ppm > 20, Abundances (grouped) CV% 0 ppm > 25, proteins with a combination of low PEP score and CV% > 15, PEP score < 7, and proteins that had a ‘Found in Sample’ value of any value lower than ‘High’ for at least 2 individuals.

Proteomics raw data can be found in Supplementary Table ³.