癌症信号蛋白的多重液相色谱-多反应监测质谱定量分析
Multiplexed Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry Quantification of Cancer Signaling Proteins.
作者信息
Chen Yi, Fisher Kate J, Lloyd Mark, Wood Elizabeth R, Coppola Domenico, Siegel Erin, Shibata David, Chen Yian A, Koomen John M
机构信息
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.
出版信息
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。
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