Stokes Matthew P, Silva Jeffrey C, Jia Xiaoying, Lee Kimberly A, Polakiewicz Roberto D, Comb Michael J
Cell Signaling Technology, 3 Trask Lane, Danvers, MA 01923, USA.
Int J Mol Sci. 2012 Dec 21;14(1):286-307. doi: 10.3390/ijms14010286.
Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA.
使用液相色谱和串联质谱（LC-MS/MS）分析肽段的传统方法，由于质谱仪器固有的 duty cycle 限制以及分析平台的随机性，缺乏全面监测特定生物学过程的特异性。PTMScan Direct 是一种基于抗体的新型方法，可对存在于同一信号通路中的蛋白质的特定肽段进行定量 LC-MS/MS 分析。现已生产出新型 PTMScan Direct 试剂，其靶向参与 DNA 损伤 / 细胞周期以及凋亡 / 自噬通路的蛋白质的肽段。这些试剂共同提供了对这些信号级联反应中 237 种蛋白质上 438 个位点的检测途径。这些试剂已用于分析人类细胞系中 DNA 对紫外线损伤的反应。如通过与切割、活化的半胱天冬酶相对应的肽段丰度增加所评估的那样，紫外线损伤显示通过 ATM/ATR 依赖性信号传导激活经典的 DNA 损伤反应通路、应激反应通路并诱导凋亡的启动。这些数据证明了 PTMScan Direct 作为一种多重分析方法用于分析细胞对各种刺激（如 DNA 的紫外线损伤）的特异性反应的实用性。
