Targeted Proteomics Analysis for Parvalbumin in Rat Brain Tissue
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Thyroid hormones (THs) modulate the differentiation and fate-specificity of inhibitory neurons in the developing brain. A subset of GABAergic inhibitory neurons expresses the low molecular weight calcium-binding protein parvalbumin (Pvalb). The presence of Pvalb confers specific firing properties upon these neurons that are essential for neuronal wiring, solidification of synaptic contacts, and neuronal plasticity. TH regulates both the maturation and timing of expression of Pvalb, and these interneurons appear to be particularly sensitive to disruption of the thyroid system, including exposure to environmental contaminants. We sought to develop methods to improve detection and facilitate quantification of perturbations of this novel biomarker of TH disruption. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a targeted proteomics approach to examine Pvalb in the cortex of the rat brain. The anterior cortex was collected from adult Long Evans rats and homogenized in a buffer containing dithiothreitol and then denatured. An in-silico tryptic digest was used to cleave Pvalb, resulting in peptides ~ 8-25 amino acids in length and free of internal lysine and arginine. Using recombinant Pvalb, in-silico digestion and LC-MS/MS confirmed that peptide fragments SGFIEEDELGSILK and IGVEEFSTLVAES were abundant in the cortex of the rat brain and contained multiple transitions for quantitation and confirmation. For quantitation, the digestant was spiked with isotope labeled Pvalb peptides, and the mixture was purified by mixed-mode solid-phase extraction prior to analysis by LC-MS/MS. These preliminary findings demonstrate analyte specificity and sensitivity for the quantification of Pvalb protein and will be applied to ongoing efforts to identify the impact of TH-disrupting chemicals on the developing brain. This work does not reflect US EPA policy.