Russo Paul, Hood Brian L, Bateman Nicholas W, Conrads Thomas P
Center for Applied Proteomics and Molecular Medicine, George Mason University, 10920 George Mason Circle MS1A9, Manassas, VA, 20110, USA.
DOD Gynecologic Cancer Center of Excellence, Annandale, VA, 22003, USA.
Methods Mol Biol. 2017;1606:313-332. doi: 10.1007/978-1-4939-6990-6_20.
Selected reaction monitoring (SRM) is used in molecular profiling to detect and quantify specific known proteins in complex mixtures. Using isotope dilution (Barnidge et al., Anal Chem 75(3):445-451, 2003) methodologies, peptides can be quantified without the need for an antibody-based method. Selected reaction monitoring assays employ electrospray ionization mass spectrometry (ESI-MS) followed by two stages of mass selection: a first stage where the mass of the peptide ion is selected and, after fragmentation by collision-induced dissociation (CID), a second stage (tandem MS) where either a single (e.g., SRM) or multiple (multiple reaction monitoring, MRM) specific peptide fragment ions are transmitted for detection. The MRM experiment is accomplished by specifying the parent masses of the selected endogenous and isotope-labeled peptides for MS/MS fragmentation and then monitoring fragment ions of interest, using their intensities/abundances and relative ratios to quantify the parent protein of interest. In this example protocol, we will utilize isotope dilution MRM-MS to quantify in absolute terms the total levels of the protein of interest, ataxia telangiectasia mutated (ATM) serine/threonine protein kinase. Ataxia telangiectasia mutated (ATM) phosphorylates several key proteins that initiate activation of the DNA damage checkpoint leading to cell cycle arrest.
选择反应监测（SRM）用于分子谱分析，以检测和定量复杂混合物中特定的已知蛋白质。采用同位素稀释法（Barnidge等人，《分析化学》75(3):445 - 451, 2003），无需基于抗体的方法即可对肽段进行定量。选择反应监测分析采用电喷雾电离质谱（ESI-MS），随后进行两个质量选择阶段：第一阶段选择肽离子的质量，在通过碰撞诱导解离（CID）进行碎片化后，第二阶段（串联质谱）传输单个（例如SRM）或多个（多反应监测，MRM）特定肽段碎片离子进行检测。MRM实验通过指定用于MS/MS碎片化的所选内源性和同位素标记肽段的母离子质量来完成，然后监测感兴趣的碎片离子，利用它们的强度/丰度和相对比率来定量感兴趣的母蛋白。在本示例方案中，我们将利用同位素稀释MRM-MS以绝对量定量感兴趣的蛋白质——共济失调毛细血管扩张症突变（ATM）丝氨酸/苏氨酸蛋白激酶的总水平。共济失调毛细血管扩张症突变（ATM）使几种关键蛋白质磷酸化，从而启动DNA损伤检查点的激活，导致细胞周期停滞。
