Delayed Times to Tissue Fixation Result in Unpredictable Global Phosphoproteome Changes

Protein phosphorylation controls the activity of signal transduction pathways regulated by kinases and phosphatases. Little is known, however, about the impact of preanalytical factors, for example, delayed times to tissue fixation, on global phosphoprotein levels in tissues. The aim of this study was to characterize the potential effects of delayed tissue preservation (cold ischemia) on the levels of phosphoproteins using targeted and nontargeted proteomic approaches. Rat and murine liver samples were exposed to different cold ischemic conditions ranging from 10 to 360 min prior to cryopreservation. The phosphoproteome was analyzed using reverse phase protein array (RPPA) technology and phosphoprotein-enriched quantitative tandem mass spectrometry (LC-MS/MS). RPPA analysis of rat liver tissues with long (up to 360 min) cold ischemia times did not reveal statistically significant alterations of specific phosphoproteins even though nonphosphorylated cytokeratin 18 (CK18) showed increased levels after 360 min of delay to freezing. Keeping the samples on ice prior to cryopreservation prevented this effect. LC-MS/MS-based quantification of 1684 phosphorylation sites in rat liver tissues showed broadening of their distribution compared to time point zero, but without reaching statistical significance for individual phosphosites. Similarly, RPPA analysis of mouse liver tissues with short (<60 min) cold ischemia times did not reveal directed or predictable changes of protein and phosphoprotein levels. Using LC-MS/MS and quantification of 791 phosphorylation sites, we found that the distribution of ratios compared to time point zero broadens with prolonged ischemia times, but these were rather undirected and diffuse changes, as we could not detect significant alterations of individual phosphosites. On the basis of our results from RPPA and LC-MS/MS analysis of rat and mouse liver tissues, we conclude that prolonged cold ischemia results in unspecific phosphoproteome changes that can be neither predicted nor assigned to individual proteins. On the other hand, we identified a number of phosphosites which were extraordinarily stable even after 360 min of cold ischemia and, therefore, may be used as general reference markers for future companion diagnostics for kinase inhibitors.蛋白质磷酸化（protein phosphorylation）调控由激酶（kinases）与磷酸酶（phosphatases）调控的信号转导通路活性。然而，目前对于分析前因素——例如组织固定延迟时长——对组织中整体磷酸化蛋白水平的影响，人们尚所知甚少。本研究旨在借助靶向与非靶向蛋白质组学方法，解析组织保存延迟（即冷缺血（cold ischemia））对磷酸化蛋白水平的潜在影响。我们将大鼠与小鼠肝脏样本在冷冻保存（cryopreservation）前，置于时长覆盖10至360分钟的不同冷缺血条件下。采用反相蛋白质阵列（reverse phase protein array, RPPA）技术与磷酸化蛋白富集定量串联质谱（液相色谱-串联质谱，LC-MS/MS）对磷酸化蛋白质组（phosphoproteome）进行分析。对冷缺血时长较长（最长达360分钟）的大鼠肝脏组织进行RPPA分析，未发现特定磷酸化蛋白存在统计学意义上的显著改变；尽管非磷酸化细胞角蛋白18（cytokeratin 18, CK18）在冷冻延迟360分钟后水平有所升高。将样本在冷冻保存前置于冰上，可阻断这一效应。针对大鼠肝脏组织中1684个磷酸化位点（phosphosites）的LC-MS/MS定量分析显示，与0时间点相比，其分布范围有所扩大，但单个磷酸化位点未达到统计学显著性差异。类似地，对冷缺血时长较短（<60分钟）的小鼠肝脏组织进行RPPA分析，未发现蛋白质与磷酸化蛋白水平出现定向或可预测的变化。通过LC-MS/MS对791个磷酸化位点进行定量后，我们发现相较于0时间点，比值的分布范围随缺血时长延长而扩大，但此类变化并无定向性且呈弥散性：我们未能检测到单个磷酸化位点存在显著改变。综合大鼠与小鼠肝脏组织的RPPA及LC-MS/MS分析结果，我们得出结论：长时间冷缺血会引发非特异性的磷酸化蛋白质组变化，此类变化既无法预测，也无法归因于单个蛋白质。另一方面，我们鉴定出多个磷酸化位点，即便在冷缺血360分钟后仍表现出极强的稳定性，因此可作为通用参考标志物，用于未来针对激酶抑制剂（kinase inhibitors）的伴随诊断（companion diagnostics）。