Gámez-Pozo Angelo, Sánchez-Navarro Iker, Calvo Enrique, Díaz Esther, Miguel-Martín María, López Rocío, Agulló Teresa, Camafeita Emilio, Espinosa Enrique, López Juan Antonio, Nistal Manuel, Vara Juan Ángel Fresno
Instituto de Investigación Sanitaria, IdIPAZ, Hospital Universitario La Paz, Madrid, Spain.
Mol Biosyst. 2011 Aug;7(8):2368-74. doi: 10.1039/c1mb05113j. Epub 2011 May 26.
Protein phosphorylation affects most eukaryotic cellular processes and its deregulation is considered a hallmark of cancer and other diseases. Phosphoproteomics may enable monitoring of altered signaling pathways as a means of stratifying tumors and facilitating the discovery of new drugs. Unfortunately, the development of molecular tests for clinical use is constrained by the limited availability of fresh frozen, clinically annotated samples. Here we report phosphopeptide analysis in human archival formalin-fixed, paraffin-embedded (FFPE) cancer samples based on immobilized metal affinity chromatography followed by liquid chromatography coupled with tandem mass spectrometry and selected reaction monitoring techniques. Our results indicate the equivalence of detectable phosphorylation rates in archival FFPE and fresh frozen tissues. Moreover, we demonstrate the applicability of targeted assays for phosphopeptide analysis in clinical archival FFPE samples, using an experimental workflow suitable for processing and analyzing large sample series. This work paves the way for the application of shotgun and targeted phosphoproteomics approaches in clinically relevant studies using archival clinical samples.
蛋白质磷酸化影响大多数真核细胞过程，其失调被认为是癌症和其他疾病的一个标志。磷酸化蛋白质组学可以监测信号通路的改变，以此作为对肿瘤进行分层和促进新药发现的一种手段。不幸的是，临床用分子检测方法的开发受到新鲜冷冻且带有临床注释样本有限的限制。在此，我们报告基于固定化金属亲和色谱，随后进行液相色谱与串联质谱联用以及选择反应监测技术，对人类存档福尔马林固定石蜡包埋（FFPE）癌症样本进行磷酸肽分析。我们的结果表明存档FFPE组织和新鲜冷冻组织中可检测到的磷酸化率相当。此外，我们使用适合处理和分析大量样本系列的实验流程，证明了靶向分析在临床存档FFPE样本中进行磷酸肽分析的适用性。这项工作为在使用存档临床样本的临床相关研究中应用鸟枪法和靶向磷酸化蛋白质组学方法铺平了道路。
