The organ perfusion solution (perfusate), collected at clinically and temporally significant stages of the organ preservation and transplantation process, provides a valuable insight into the biological status of an organ over time and prior to reperfusion (transplantation) in the recipient. The objective of this study was to assess two bottom-up proteomics workflows for the extraction of tryptic peptides from the perfusate. Two different kinds of perfusate samples from kidney and liver trials were profiled using liquid chromatography–mass spectrometry (LC-MS/MS). The preparation of clean peptide mixtures for downstream analysis was performed considering different aspects of sample preparation; protein estimation, enrichment, in-gel and urea-based in-solution digestion. In-solution digestion of perfusate allowed identification of the highest number of peptides and proteins with greater sequence coverage and higher confidence data in kidney and liver perfusate. Key pathways identified by gene ontology analysis included complement, coagulation and antioxidant pathways, and a number of biomarkers previously linked to ischemia-reperfusion injury were also observed in perfusate. This study showed that in-solution digestion is a more efficient method for LC-MS/MS analysis of kidney and liver organ perfusion solutions. This method is also quicker and easier than in-gel digestion, allowing for greater sample throughput, with fewer opportunities for experimental error or peptide loss.

中文翻译：

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器官灌注溶液蛋白质组分析中凝胶内和溶液内蛋白水解的比较

在器官保存和移植过程的临床和时间重要阶段收集的器官灌注溶液（灌注液）可以为了解器官随时间变化以及在受体再灌注（移植）之前的生物学状态提供有价值的见解。本研究的目的是评估两种从灌注液中提取胰蛋白酶肽的自下而上的蛋白质组学工作流程。使用液相色谱-质谱法 (LC-MS/MS) 对来自肾脏和肝脏试验的两种不同类型的灌注液样品进行了分析。考虑到样品制备的不同方面，制备用于下游分析的清洁肽混合物；蛋白质估计、富集、凝胶内和基于尿素的溶液内消化。灌流液的溶液内消化可以鉴定最多数量的肽和蛋白质，并且在肾脏和肝脏灌流液中具有更大的序列覆盖度和更高的置信度数据。通过基因本体分析确定的关键途径包括补体、凝血和抗氧化途径，并且在灌注液中还观察到了许多先前与缺血再灌注损伤相关的生物标志物。本研究表明，溶液内消化是对肾脏和肝脏器官灌注溶液进行 LC-MS/MS 分析的更有效方法。该方法比凝胶内消化更快、更容易，可实现更大的样品通量，同时减少实验错误或肽损失的机会。