TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis.
TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis.
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DOI：
10.3791/60970
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发表时间：
2020-06
期刊：
影响因子：
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通讯作者：
Silveli Suzuki-Hatano;A. Tsai;Audrey L. Daugherty;C. A. Pacak
中科院分区：
文献类型：
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作者：
Silveli Suzuki-Hatano;A. Tsai;Audrey L. Daugherty;C. A. Pacak
蛋白质组技术是一种强大的方法，可以通过提供疾病、治疗或其他条件对整个蛋白质组影响的全局视图来帮助我们理解生物系统中的作用机制。该报告提供了蛋白质样品的提取、定量、沉淀、消化、标记和后续数据分析的详细方案。我们优化的 TMT 标记方案需要较低的标签浓度，并获得一致可靠的数据。我们使用该方案来评估各种小鼠组织（即心脏、骨骼肌和大脑）以及体外培养的细胞中的蛋白质表达谱。此外，我们还演示了如何从生成的数据集中评估数千种蛋白质。
Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.