We investigated the effect of homogenization strategy and protein precipitation on downstream protein quantitation using multiple reaction monitoring mass spectrometry (MRM-MS). Our objective was to develop a workflow capable of processing disparate tissue types with high throughput, minimal variability, and maximum purity. Similar abundances of endogenous proteins were measured in nine different mouse tissues regardless of the homogenization method used; however, protein precipitation had strong positive effects on several targets. The best throughput was achieved by lyophilizing tissues to dryness, followed by homogenization via bead-beating without sample buffer. Finally, the effect of tissue perfusion prior to dissection and collection was explored in 20 mouse tissues. MRM-MS showed decreased abundances of blood-related proteins in perfused tissues; however, complete removal was not achieved. Concentrations of nonblood proteins were largely unchanged, although significantly higher variances were observed for proteins from the perfused lung, indicating that perfusion may not be suitable for this organ. We present a simple yet effective tissue processing workflow consisting of harvest of fresh nonperfused tissue, novel lyophilization and homogenization by bead-beating, and protein precipitation. This workflow can be applied to a range of mouse tissues with the advantages of simplicity, minimal manual manipulation of samples, use of commonly available equipment, and high sample quality.

中文翻译：

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制备用于蛋白质组学分析的不同小鼠组织的过程和工作流程

我们使用多个反应监测质谱（MRM-MS），研究了均质化策略和蛋白质沉淀对下游蛋白质定量的影响。我们的目标是开发一种工作流程，该流程能够以高通量，最小的变异性和最大的纯度处理不同类型的组织。无论使用何种均质方法，都在九种不同的小鼠组织中测量了相似的内源蛋白丰度；然而，蛋白质沉淀对多个靶标具有很强的积极作用。通过将组织冻干至干，然后通过无需样品缓冲液的打珠。最后，在20个小鼠组织中研究了解剖和收集之前组织灌注的效果。MRM-MS显示灌注组织中血液相关蛋白的丰度降低；但是，无法完全清除。非血液蛋白质的浓度基本没有变化，尽管从灌注肺中观察到的蛋白质差异明显更高，这表明灌注可能不适用于该器官。我们提出了一个简单而有效的组织处理工作流程，包括收获新鲜的非灌注组织，新型冻干和通过磁珠打浆进行均质化以及蛋白质沉淀。该工作流程可应用于多种小鼠组织，其优点是简单，样品的手动操作最少，使用常用设备，