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Neuroinflammation is a hallmark of both acute and chronic neurodegenerative diseases and is driven, in part, by activated glial cells, including microglia. A key regulator of this inflammatory response is the NLRP3 inflammasome, an immune sensor that can be triggered by diverse, unrelated stimuli such as pathogen-associated molecular patterns, cellular stress, and mitochondrial dysfunction. Despite progress in targeting NLRP3-mediated immune activation, many drug candidates fail, potentially due to the limited availability of physiologically relevant disease models. The SIM-A9 murine microglial cell line, established in 2014, has emerged as a widely used model for studying neuroinflammation; however, its proteome has not yet been systematically characterized. In this study, we investigated the proteomic landscape of SIM-A9 microglia treated with classical pro-inflammatory stimuli, including lipopolysaccharide (LPS) and extracellular ATP and nigericin (NG), to induce NLRP3 inflammasome activation. Using complementary proteomic approaches, we quantified 4903 proteins and observed significant enrichment of proteins associated with immune and nervous system processes. Differentially expressed proteins were consistent with an activated microglial phenotype, including the upregulation of proteins involved in NLRP3 inflammasome signaling. To our knowledge, this is the first comprehensive proteomic analysis of SIM-A9 microglia. These findings provide a foundational resource that may enhance the interpretation and design of future studies using SIM-A9 cells as a model of neuroinflammation. Full article

Mass spectrometry is a powerful technique for investigating renal pathologies and identifying biomarkers, and efficient protein extraction from kidney tissue is essential for bottom-up proteomic analyses. Detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible with downstream protein digestion and liquid chromatography-coupled mass spectrometry, requiring additional purification and buffer-exchange steps. This study compares two well-established detergent-based methods for protein extraction (in-solution sodium deoxycholate (SDC); suspension trapping (S-Trap)) with the recently developed sample preparation by easy extraction and digestion (SPEED) method, which uses strong acid for denaturation. We compared the quantitative performance of each method using label-free mass spectrometry in both sheep kidney cortical tissue and plasma. In kidney tissue, SPEED quantified the most unique proteins (SPEED 1250; S-Trap 1202; SDC 1197). In plasma, S-Trap produced the most unique protein quantifications (S-Trap 150; SDC 148; SPEED 137). Protein quantifications were reproducible across biological replicates in both tissue (R² = 0.85–0.90) and plasma (SPEED R² = 0.84; SDC R² = 0.76, S-Trap R² = 0.65). Our data suggest SPEED as the optimal method for proteomic preparation in kidney tissue and S-Trap or SPEED as the optimal method for plasma, depending on whether a higher number of protein quantifications or greater reproducibility is desired. Full article