Project description:Label free quantititative phosphoproteomics analysis following TiO2 enrichment, nanoscale capillary chromatography and high resolution tandem mass spectrometry.
Project description:This study aims to investigate the protein expression profiles in a murine model of dextran sulfate sodium (DSS)-induced colitis using advanced Astral-DIA quantitative proteomics technology. A total of 12 colon tissue samples were analyzed, including 6 from healthy control mice and 6 from DSS-treated mice with induced colitis. Experimental Design Species: Mus musculus (C57BL/6 strain). Tissue Source: Colon tissues were dissected, snap-frozen in liquid nitrogen, and homogenized to extract proteins. Groups: Control Group: Healthy mice without intervention. DSS Group: Mice subjected to 2.5% DSS administration for 7 days to induce colitis, validated by histopathological assessment.
Project description:Toll/interleukin-1 receptor (TIR) domain proteins are immune signaling components and function as NAD+-cleaving enzymes to activate defense responses. Activation of TIRs represses growth and drives cell death in plants and promotes axon degeneration in animals, but how plant TIRs are repressed remains unclear. Here, we show that TIR NADase activity requires a conserved serine residue spatially close to the catalytic glutamate. The plant Ca2+-dependent protein kinases (CPKs), the mammalian Ca2+/calmodulin-dependent protein kinase II delta (CAMK2D) and TANK binding kinase 1 (TBK1) phosphorylate TIR domains at this conserved serine, which blocks TIR NADase activities and functions and thus maintains growth in plants and suppresses SARM1 TIR signaling in animals, respectively. Our findings define a fundamental molecular mechanism by which phosphorylation at a conserved serine residue blocks TIR signaling to balance growth and defense trade-offs.
Project description:Lysosomes are implicated in a wide spectrum of human diseases including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration and cancer. Profiling lysosomal content using tag-based lysosomal immunopurification (LysoIP) in cell and animal models allowed major discoveries in the field, however, studying lysosomal dysfunction in human patients remains a challenge. Here, we report the development of the tagless LysoIP method to enable rapid enrichment of lysosomes, via immunoisolation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cells. Isolated lysosomes are intact and suitable for subsequent multimodal omics analyses. To validate the utility of our approach, we employed the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells (PBMCs) derived from fresh blood of patients with CLN3 Batten disease, a neurodegenerative LSD. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify novel biomarkers and discover human-relevant disease mechanisms.
Project description:Chronic pain substantially affects the mental and physical well-being of patients and magnifies the socio-economic burden on the healthcare system. It is important to understand the molecular mechanisms underlying chronic pain to effectively target it. To investigate peripheral mechanisms relevant to pain signaling, we isolated nerve terminals from mouse footpads. The isolated peripheral terminals contain both pre- and post-synaptic proteins and are deficient in keratin and histone in both mice and humans. We detected the protein translational machinery and mitochondria in nerve terminals and observed that they were capable of endocytosis. An unbiased proteomic analysis of nerve terminals from footpads of NaV1.9 knockout mice shows dysregulation of the p38 mitogen-activated protein kinase (MAPK) and extracellular regulated kinase 1/2 (ERK1/2) pathways, and of protein components involved in translation and energy metabolism. Our study thus reveals peripheral signaling mechanisms implicated in pain perception.
Project description:The circadian clock drives daily rhythms of gene expression and physiological functions across tissues throughout the body. Advances in next-generation DNA sequencing have provided extensive insights into gene expression at the RNA level, but more functional information at the protein level with sufficient depth has been limited by technical challenges. Recently, the next-generation mass spectrometer Orbitrap Astral was developed, allowing us to quantify protein abundance with greater sensitivity and accuracy. In this study, we generated a comprehensive mouse circadian proteome atlas (available in https://chiba1.dynacom.co.jp/mcp_atlas/) by analyzing protein abundance in 32 tissues including the suprachiasmatic nucleus (SCN) across the day using Orbitrap Astral. Data-independent acquisition of 584 samples including developmental samples revealed the spatiotemporal profiles of 18,956 proteins, accounting for 73.8% of all proteins registered in UniProt. Proteome and phospho-proteome analyses of whole-cell and nuclear proteins in the liver uncovered circadian profiles not only in protein abundance but also in subcellular localization and post-translational modification. Notably, proteome analysis revealed global changes in protein phosphorylation status in hPER2-S662G mutant mice, a genetic model of human familial advanced sleep phase (FASP). This multi-tissue circadian proteome atlas provides a fundamental resource for understanding when, where, and which proteins are expressed and function.
Project description:The circadian clock drives daily rhythms of gene expression and physiological functions across tissues throughout the body. Advances in next-generation DNA sequencing have provided extensive insights into gene expression at the RNA level, but more functional information at the protein level with sufficient depth has been limited by technical challenges. Recently, the next-generation mass spectrometer Orbitrap Astral was developed, allowing us to quantify protein abundance with greater sensitivity and accuracy. In this study, we generated a comprehensive mouse circadian proteome atlas (available in https://chiba1.dynacom.co.jp/mcp_atlas/) by analyzing protein abundance in 32 tissues including the suprachiasmatic nucleus (SCN) across the day using Orbitrap Astral. Data-independent acquisition of 584 samples including developmental samples revealed the spatiotemporal profiles of 18,956 proteins, accounting for 73.8% of all proteins registered in UniProt. Proteome and phospho-proteome analyses of whole-cell and nuclear proteins in the liver uncovered circadian profiles not only in protein abundance but also in subcellular localization and post-translational modification. Notably, proteome analysis revealed global changes in protein phosphorylation status in hPER2-S662G mutant mice, a genetic model of human familial advanced sleep phase (FASP). This multi-tissue circadian proteome atlas provides a fundamental resource for understanding when, where, and which proteins are expressed and function.