Site-specific detection of S-nitrosylated PKB α/Akt1 from rat soleus muscle using CapLC-Q-TOFmicro mass spectrometry
Site-specific detection of S-nitrosylated PKB α/Akt1 from rat soleus muscle using CapLC-Q-TOFmicro mass spectrometry
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DOI：
10.1002/jms.885
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发表时间：
2005-09-01
影响因子：
2.3
通讯作者：
Fischman, AJ
中科院分区：
文献类型：
--
作者：
Lu, XM;Lu, M;Fischman, AJ
蛋白激酶 B α（PKB α 或 Akt1）被认为在程序性细胞死亡、癌症进展和胰岛素信号级联中发挥着至关重要的作用。该蛋白通过多个位点的磷酸化被激活，随后磷酸化并激活 eNOS。蛋白质的游离半胱氨酸残基可以捕获反应性的、内源性产生的一氧化氮 (NO) 作为 S-亚硝基硫醇。 S-亚硝基化半胱氨酸残基的位点特异性检测（通常在低化学计量下）一直是蛋白质组学研究中的主要挑战，这主要是由于缺乏在生理条件下非常不稳定的 S-亚硝基硫醇的质量标记。在本报告中，我们描述了一种灵敏且特异的 MS 方法，用于检测大鼠比目鱼肌中 PKB α/Akt1 中的 S-亚硝基硫醇。PKB a/Akt1 通过免疫沉淀和 2D 凝胶电泳分离，进行凝胶内胰蛋白酶消化，半胱氨酰亚硝基硫醇在抗坏血酸还原下与碘乙酸 [2-C-12/C-13 = 50/50] 反应条件。这导致在高 MS 噪声区域的窄氩碰撞能量 (CE = 30 +/- 5 V) 内产生相对稳定的羧甲基半胱氨酸 (CMC) 亚铵离子（m/z 134.019 和 m/z 135.019）。此外，游离的和二硫键连接的半胱氨酸残基被转化为羧氨基甲基半胱氨酸（CAM）。在毛细管液相色谱 (LC) 分离过程中，在触发洗脱时间，以 50 mDa 的质量精度检测胰蛋白酶 S-亚硝基化母离子。在锁定质量条件 (m/z 785.8426) 下，将含有 Cys(296) 的肽与四种共洗脱胰蛋白酶肽区分开来。胰蛋白酶肽 ITDFGLBKEGIK（B：CAM，[M + 2H](2+) = 690.86，发现值：690.83）中的 S-亚硝基硫醇被认为以非常低的水平存在，因为在 MS 调查中 CMC 铵触发离子的阈值设置为 3 计数/秒。在应激条件下产生的高水平 NO 可能导致 S-亚硝基化增加Cys(216) 可阻断 Cys(296) 和 Cys(310) 之间二硫键的形成，并抑制 PKB α/Akt1 的生物效应。通过这里开发的程序，可以在生理和病理条件下研究这个过程。版权所有 (c) 2005 John Wiley & Sons, Ltd.
Protein Kinase B alpha(PKB alpha, or Akt1) is believed to play a crucial role in programmed cell death, cancer progression and the insulin-signaling cascade. The protein is activated by phosphorylation at multiple sites and subsequently phosphorylates and activates eNOS. Free cysteine residues of the protein may capture reactive, endogenously produced nitric oxide (NO) as S-nitrosothiols. Site-specific detection of S-nitrosylated cysteine residues, usually at low stoichiometry, has been a major challenge in proteomic research largely due to the lack of mass marker for S-nitrosothiols that are very labile under physiologic conditions. In this report we describe a sensitive and specific MS method for detection of S-nitrosothiols in PKB alpha/Akt1 in rat soleus muscle.PKB a/Akt1 was isolated by immunoprecipitation and 2D-gel electrophoresis, subjected to in-gel tryptic digestion, and cysteinyl nitrosothiols were reacted with iodoacetic acids [2-C-12/C-13 = 50/50] under ascorbate reduction conditions. This resulted in the production of relatively stable carboxymethylcysteine (CMC) immonium ions (m/z 134.019 and m/z 135.019) within a narrow argon collision energy (CE = 30 +/- 5 V) in the high MS noise region. In addition, free and disulfide-linked cysteine residues were converted to carboxyamiclomethylcysteines (CAM). Tryptic S-nitrosylated parent ion was detected with a mass accuracy of 50 mDa for the two CMC immonium ions at the triggered elution time during capillary liquid chromatography (LC) separation. A peptide containing Cys(296) was discriminated from four co-eluting tryptic peptides under lock mass conditions (m/z 785.8426). S-nitrosothiol in the tryptic peptide, ITDFGLBKEGIK (B: CAM, [M + 2H](2+) = 690.86, Found: 690.83), is believed to be present at a very low level, since the threshold for the CMC immonium trigger ions was set at 3 counts/s in the MS survey.The high levels of NO that are produced under stress conditions may result in increased S-nitrosylation Of Cys(216) which blocks disulfide bond formation between Cys(296) and Cys(310) and suppresses the biological effects of PKB alpha/Akt1. With the procedures developed here, this process can be studied under physiological and pathological conditions. Copyright (c) 2005 John Wiley & Sons, Ltd.