Chen Yi, Fisher Kate J, Lloyd Mark, Wood Elizabeth R, Coppola Domenico, Siegel Erin, Shibata David, Chen Yian A, Koomen John M
H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL, USA.
Department of Surgery, University of Tennessee Health Science Center, Memphis, TN, USA.
Methods Mol Biol. 2017;1647:19-45. doi: 10.1007/978-1-4939-7201-2_2.
Quantitative evaluation of protein expression across multiple cancer-related signaling pathways (e.g., Wnt/β-catenin, TGF-β, receptor tyrosine kinases (RTK), MAP kinases, NF-κB, and apoptosis) in tumor tissues may enable the development of a molecular profile for each individual tumor that can aid in the selection of appropriate targeted cancer therapies. Here, we describe the development of a broadly applicable protocol to develop and implement quantitative mass spectrometry assays using cell line models and frozen tissue specimens from colon cancer patients. Cell lines are used to develop peptide-based assays for protein quantification, which are incorporated into a method based on SDS-PAGE protein fractionation, in-gel digestion, and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM/MS). This analytical platform is then applied to frozen tumor tissues. This protocol can be broadly applied to the study of human disease using multiplexed LC-MRM assays.
对肿瘤组织中多种癌症相关信号通路（如Wnt/β-连环蛋白、转化生长因子-β、受体酪氨酸激酶（RTK）、丝裂原活化蛋白激酶、核因子κB和凋亡相关通路）的蛋白质表达进行定量评估，可能有助于为每个肿瘤个体建立分子图谱，从而辅助选择合适的靶向癌症治疗方法。在此，我们描述了一种广泛适用的方案，该方案利用细胞系模型和结肠癌患者的冷冻组织标本，开发并实施定量质谱分析。细胞系用于开发基于肽段的蛋白质定量分析方法，该方法被整合到一种基于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）蛋白质分级分离、胶内酶解和液相色谱-多反应监测质谱（LC-MRM/MS）的方法中。然后将该分析平台应用于冷冻肿瘤组织。该方案可广泛应用于使用多重LC-MRM分析的人类疾病研究。
