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首页> 外文期刊>Circulation research: a journal of the American Heart Association >Measurement of S-nitrosylation occupancy in the myocardium with cysteine-reactive tandem mass tags: Short communication
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Measurement of S-nitrosylation occupancy in the myocardium with cysteine-reactive tandem mass tags: Short communication

机译：半胱氨酸反应性串联质量标签测量心肌中S-亚硝基化的占有率：短时通信

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RATIONALE: S-nitrosylation (SNO) is a reversible, thiol-based protein modification that plays an important role in the myocardium by protecting critical cysteine residues from oxidation. However, little is known with regard to the percentage of a given protein that is modified by SNO (ie, SNO occupancy). Current methods allow for the relative quantification of SNO levels, but not for the determination of SNO occupancy. OBJECTIVE: To develop a method for the measurement of SNO occupancy, and apply this methodology to determine SNO occupancy in the myocardium. METHODS AND RESULTS: We developed a differential cysteinereactive tandem mass tag (cysTMT) labeling procedure for the measurement of SNO occupancy. To validate this cysTMT labeling method, we treated whole-heart homogenates with the S-nitrosylating agent S-nitrosoglutathione and determined maximal SNO occupancy. We also examined SNO occupancy under more physiological conditions and observed that SNO occupancy is low for most protein targets at baseline. Following ischemic preconditioning, SNO occupancy increased to an intermediate level compared to baseline and Snitrosoglutathione treatment, and this is consistent with the ability of SNO to protect against cysteine oxidation. CONCLUSIONS: This novel cysTMT labeling approach provides a method for examining SNO occupancy in the myocardium. Using this approach, we demonstrated that IPC-induced SNO occupancy levels are sufficient to protect against oxidation.
机译：理由：S-亚硝基化（SNO）是一种可逆的，基于硫醇的蛋白质修饰，通过保护关键的半胱氨酸残基不被氧化，在心肌中起重要作用。但是，关于由SNO修饰的给定蛋白质的百分比（即SNO占用率）知之甚少。当前的方法允许对SNO水平进行相对定量，但不能确定SNO占用率。目的：开发一种测量SNO占有率的方法，并将其应用于确定心肌中SNO占有率的方法。方法和结果：我们开发了一个半胱氨酸活性串联质量标签（cysTMT）标记程序，用于测量SNO的占有率。为了验证这种cysTMT标记方法，我们用S-亚硝基化剂S-亚硝基谷胱甘肽处理了全心脏匀浆，并确定了最大SNO占用率。我们还检查了更多生理条件下的SNO占用率，并观察到基线时大多数蛋白质目标的SNO占用率较低。缺血预处理后，与基线和亚硝基谷胱甘肽治疗相比，SNO的占有率增加到中等水平，这与SNO防止半胱氨酸氧化的能力相一致。结论：这种新颖的cysTMT标记方法提供了一种检查心肌中SNO占用的方法。使用这种方法，我们证明了IPC诱导的SNO占用水平足以防止氧化。

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