Preserving protein profiles in tissue samples: Differing outcomes with and without heat stabilization
Preserving protein profiles in tissue samples: Differing outcomes with and without heat stabilization
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DOI：
10.1016/j.jneumeth.2011.01.004
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发表时间：
2011-03-15
影响因子：
3
通讯作者：
Gardiner, Katheleen J.
中科院分区：
文献类型：
--
作者：
Ahmed, Md Mahiuddin;Gardiner, Katheleen J.
蛋白质的翻译后修饰(PTM)和蛋白质降解处理有助于调节它们的稳定性、细胞内定位和与其他蛋白质的相互作用，并直接增强或抑制它们的活性。PTM和蛋白质分解都是动态的；水平和速率都会随着细胞环境的变化而变化。组织切除、死后间隔以及随后用于蛋白质分析的组织处理方法不可避免地改变了细胞环境，从而改变了蛋白质谱的特征。为了帮助理解这些变化的时间框架和蛋白质特异性以及对它们的生物学和技术贡献，我们比较了三种组织处理方法下小鼠海马区和皮质中蛋白质谱的特征：立即裂解物制备，以及在裂解物制备之前，快速加热到95摄氏度和在液氮中标准的快速冷冻。尽管所涉及的时间框架非常短，但我们观察到蛋白质特异性的磷酸化水平的差异，总和模式的差异，以及钙调神经磷酸酶和神经营养素受体TrkA蛋白水解性切割水平的特异性差异。这些差异随着大脑区域和切除后处理时间的不同而不同，并突显了准确分析体内蛋白质组所固有的挑战。(C)2011爱思唯尔B.V.保留所有权利。
Post translational modification (PTM) and proteolytic processing of proteins contributes to regulation of their stability, intracellular localization and interactions with other proteins, and to direct enhancement or repression of their activity. Both PTM and proteolysis are dynamic; levels and rates change in response to changes in the cellular environment. Tissue excision, post mortem interval and subsequent methods of tissue processing for protein analysis unavoidably alter the cellular environment and therefore features of protein profiles. To aid in understanding the time frame and protein specificity of these changes and the biological and technical contributions to them, we have compared features of protein profiles in mouse hippocampus and cortex following three methods of tissue handling: immediate lysate preparation, and rapid heating to 95 degrees C and standard snap freezing in liquid nitrogen, prior to lysate preparation. In spite of the very short time frames involved, we observe protein-specific differences in levels of phosphorylation, general differences in patterns of sumoylation, and specific differences in levels of proteolytic cleavage of calcineurin and the neurotrophin receptor, TRKA. These differences vary with brain region and with post excision time to processing, and highlight the challenges inherent in accurately profiling the in vivo proteome. (C) 2011 Elsevier B.V. All rights reserved.