Laboratory of Cardiac Physiology, Systems Biology Center, National Heart Lung and Blood Institute, Bethesda, MD 20892, USA.
Circ Res. 2012 Oct 26;111(10):1308-12. doi: 10.1161/CIRCRESAHA.112.271320. Epub 2012 Aug 3.
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.
To develop a method for the measurement of SNO occupancy, and apply this methodology to determine SNO occupancy in the myocardium.
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.
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.
S-亚硝基化（SNO）是一种可逆的、基于巯基的蛋白质修饰，通过保护关键半胱氨酸残基免受氧化，在心肌中发挥重要作用。然而，对于特定蛋白质被 SNO 修饰的百分比（即 SNO 占有率）知之甚少。目前的方法允许相对定量 SNO 水平，但不能确定 SNO 占有率。
开发一种测量 SNO 占有率的方法，并应用该方法测定心肌中的 SNO 占有率。
我们开发了一种用于测量 SNO 占有率的差异半胱氨酸反应性串联质量标签（cysTMT）标记程序。为了验证这种 cysTMT 标记方法，我们用 S-亚硝酰谷胱甘肽处理全心脏匀浆，并确定最大 SNO 占有率。我们还在更生理的条件下检查了 SNO 占有率，观察到大多数蛋白质靶标在基线时 SNO 占有率较低。与基线和 S-nitrosoglutathione 处理相比，缺血预处理后 SNO 占有率增加到中间水平，这与 SNO 保护半胱氨酸氧化的能力一致。
这种新的 cysTMT 标记方法为研究心肌中的 SNO 占有率提供了一种方法。使用这种方法，我们证明 IPC 诱导的 SNO 占有率足以防止氧化。
