High-Throughput LC–MS/MS Proteomic Analysis of a Mouse Model of Mesiotemporal Lobe Epilepsy Predicts Microglial Activation Underlying Disease Development

Abstract

Uncovering the molecular mechanisms of Mesial temporal lobe epilepsy (MTLE) is critical to identify therapeutic targets. In this study, we performed global protein expression analysis of a kainic acid (KA) MTLE mouse model at various time-points (1d, 3d, 30d post KA injection -dpi), representing specific stages of the syndrome.High resolution liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), in combination to label-free protein quantification, using three processing approaches for quantification, was applied. Following comparison of KA versus NaCl-injected mice, 22, 53 and 175 proteins were differentially (statistically significant) expressed at 1, 3 and 30dpi respectively, according to all 3 quantification approaches. Selected findings were confirmed by multiple reaction monitoring LC-MS/MS. As a positive control, the astrocyte marker GFAP was found to be upregulated (3dpi:1.9 fold; 30dpi:12.5 fold), also verified by IHC. The results collectively suggest that impairment in synaptic transmission occurs even right after initial status epilepticus (1dpi), with neurodegeneration becoming more extensive during epileptogenesis (3dpi) and sustained at the chronic phase (30dpi), where also extensive glial and astrocyte-mediated inflammation is evident. This molecular profile is in line with observed phenotypic changes in human MTLE, providing the basis for future studies on new molecular targets for the disease.

Multiplexed MRM-based protein quantification of putative prognostic biomarkers for chronic kidney disease progression in plasma

“…Equal volume (2 μL) of plasma samples containing approximately 100 μg of total protein were used for LC-MRM-MS analysis as previously described 89,90 . Briefly, after protein denaturation (8 M urea), reduction (10 mM dithioerythritol) and alkylation (50 mM iodoacetamide) the samples were digested with trypsin [(1:100 enzyme: protein ratio (w/w)] for 16 hours in the dark (RT).…”

The impact of postsynaptic density 95 blocking peptide (Tat‐NR2B9c) and an iNOS inhibitor (1400W) on proteomic profile of the hippocampus in C57BL/6J mouse model of kainate‐induced epileptogenesis

“…Previous studies have provided important insights into the underlying cellular and molecular processes of epileptogenesis, highlighting neuroinflammation, the innate immune response, microglial activation, synaptic reorganization, and oxidative stress as key mechanisms in this regard (Bitsika et al, ; Keck et al, , ; Li et al, ; Liu et al, ; Marques‐Carneiro et al, ; Walker et al, ). Many of these observations are unsurprising, given the burgeoning knowledge base surrounding the mechanisms and pathways believed to contribute to the development of epilepsy (Aronica et al, ; Becker, ; Godale & Danzer, ; Lukawski et al, ; Pitkänen et al, ).…”

Bioinformatics and Drug Discovery

“…Bioinformatic tools used for proteomic data analysis is similar to those in transcriptomic data, i.e., using proteomic data for phenotypic screening and for drug target discovery. Most proteomic data are used in comparisons either between treatment and control animals [174][175][176] or between patients and matched normal control [177]. For example, caffeinetreated rats differ in protein expression from control rats [175].…”