Based on their potential to analyze aberrant cellular signaling in relation to biological function, kinase activity profiling in tumor biopsies by peptide microarrays and mass spectrometry-based phosphoproteomics may guide selection of protein kinase inhibitors in patients with cancer. Variable tissue handling procedures in clinical practice may influence protein phosphorylation status and kinase activity and therewith may hamper biomarker discovery. Here, the effect of cold ischemia time (CIT) on the stability of kinase activity and protein phosphorylation status in fresh-frozen clinical tissue samples was studied using peptide microarrays and mass spectrometry-based phosphoproteomics. Biopsies of colorectal cancer resection specimens from five patients were collected and snap frozen immediately after surgery and at 6 additional time points between 0 and 180 min of CIT. Kinase activity profiling was performed for all samples using a peptide microarray. MS-based global phosphoproteomics was performed in tumors from 3 patients at 4 time points. Statistical and cluster analyses were performed to analyze changes in kinase activity and phosphoproteome resulting from CIT. Unsupervised cluster analysis of kinase activity and phosphoproteome data revealed that samples from the same patients cluster together. Continuous ANOVA analysis of all 7 time points for 5 patient samples resulted in 4 peptides out of 210 (2%) with significantly (p < 0.01 and fold change > 2) altered signal intensity in time. In 4 out of 5 patients tumor kinase activity was stable with CIT. MS-based phosphoproteomics resulted in the detection of 10,488 different phosphopeptides with on average 6044 phosphopeptides per tumor sample. 2715 phosphopeptides were detected in all samples at time point 0, of which 90 (3.3%) phosphopeptides showed significant changes in intensity with CIT (p < 0.01). Only two phosphopeptides were significantly changed in all time points, including one peptide (PKP3) with a fold change > 2. The vast majority of the phosphoproteome as well as the activity of protein kinases in colorectal cancer resection tissue is stable up to 180 min of CIT and reflects tumor characteristics. However, specific changes in kinase activity with increasing CIT were observed. Therefore, stringent tissue collection procedures are advised to minimize changes in kinase activity during CIT.

中文翻译：

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冷缺血对患者新鲜冷冻结直肠癌组织中磷酸化蛋白质组和蛋白激酶活性的时间依赖性

基于它们分析与生物学功能有关的异常细胞信号转导的潜力，通过肽微阵列和基于质谱的磷酸化蛋白质组学在肿瘤活检中的激酶活性分析可指导癌症患者中蛋白激酶抑制剂的选择。临床实践中可变的组织处理程序可能影响蛋白质的磷酸化状态和激酶活性，并因此可能阻碍生物标志物的发现。在这里，使用肽微阵列和基于质谱的磷酸化蛋白质组学研究了冷缺血时间（CIT）对新鲜冷冻临床组织样品中激酶活性和蛋白质磷酸化状态的稳定性的影响。收集了来自五名患者的结直肠癌切除标本的活检样本，并在手术后立即在CIT 0至180分钟之间的另外6个时间点进行了速冻。使用肽微阵列对所有样品进行激酶活性分析。在4个时间点对3例患者的肿瘤进行了基于MS的全球磷酸化蛋白质组学研究。进行统计和聚类分析以分析由CIT引起的激酶活性和磷酸化蛋白质组的变化。激酶活性和磷酸化蛋白质组数据的无监督聚类分析表明，来自同一患者的样本聚在一起。对5个患者样品的所有7个时间点进行连续ANOVA分析，结果发现210种肽中有4种（2％）随时间变化了明显的信号强度（p <0.01和fold change> 2）。五分之四的患者中，CIT使肿瘤激酶活性稳定。基于MS的磷酸化蛋白质组学可检测到10,488种不同的磷酸化肽，每个肿瘤样本平均检测到6044种磷酸化肽。在时间点0，在所有样品中检测到2715个磷酸肽，其中90个（3.3％）磷酸肽显示CIT强度显着变化（p <0.01）。在所有时间点上，只有两种磷酸肽发生了显着变化，其中一种肽（PKP3）的变化倍数大于2。结直肠癌切除组织中的绝大多数磷酸化蛋白质组以及蛋白激酶的活性在长达180分钟的时间内都是稳定的。 CIT并反映肿瘤特征。但是，观察到激酶活性随CIT增加而发生特定变化。所以，