Assessment of Protein Carbonylation and Protein Tyrosine Phosphatase (PTP) Oxidation in Vascular Smooth Muscle Cells (VSMCs) Using Immunoblotting Approaches.
Assessment of Protein Carbonylation and Protein Tyrosine Phosphatase (PTP) Oxidation in Vascular Smooth Muscle Cells (VSMCs) Using Immunoblotting Approaches.
复制标题
使用免疫印迹方法评估血管平滑肌细胞 (VSMC) 中的蛋白质羰基化和蛋白质酪氨酸磷酸酶 (PTP) 氧化。
作者：
S. Tsiropoulou;R. Touyz
蛋白质的翻译后修饰（例如磷酸化和氧化）通过影响蛋白质的结构和功能在细胞信号传导中发挥重要作用。在血管细胞中，除了影响磷酸化外，血管紧张素 II (Ang II) 还会诱导蛋白质氧化，这对于心血管和肾脏系统的氧化还原信号传导非常重要。本章介绍了用于评估血管平滑肌细胞 (VSMC) 蛋白质组中不可逆蛋白质羰基化和蛋白质酪氨酸磷酸酶 (PTP) 氧化状态的免疫印迹方法。蛋白质羰基化通常使用 OxyBlot™ 方法进行测量，其中二硝基苯肼 (DNPH) 对氧化氨基酸上的蛋白质羰基 (C = O) 进行衍生化，从而形成稳定的二硝基苯基 (DNP)腙产品。通过 SDS-PAGE 分析样品，并使用针对 DNP 部分产生的一抗通过免疫印迹确定样品中不可逆蛋白质羰基化的水平。可以使用针对这些酶的“过氧化”(SO3H) 催化位点的单克隆抗体来评估 PTP 的氧化。所描述的方法能够区分不可逆 (SO3H) 和可逆 (SOH) PTP 氧化态。最初，游离的未修饰 PTP-硫醇 (S-) 被烷基化，并将样品分成两部分。一部分用于评估 PTP-SO3H 形式。在另一部分中，可逆修饰的 PTP-硫醇首先被还原，然后被过钒酸盐 (PV) 过度氧化。未经处理和 PV 处理的样品均通过 SDS-PAGE 进行分析，并通过免疫印迹检测“过氧化”PTP。可逆氧化的 PTP-SOH 部分的比例由未处理和 PV 处理的样品中的信号之间的差异决定。上述免疫测定提供了检测和量化任何（病理）生理背景下不可逆蛋白质氧化和不可逆/可逆氧化 PTP 的整体水平的通用方法。总体氧化还原状态的表征对于更好地了解与氧化应激相关的慢性疾病潜在的氧化还原敏感机制至关重要。这对于受肾素血管紧张素系统影响的系统尤其重要，因为 Ang II 是氧化应激和氧化还原信号传导的有效诱导剂。
Post-translational modification of proteins, such as phosphorylation and oxidation, plays a major role in cellular signaling by influencing protein structure and function. In vascular cells, in addition to influencing phosphorylation, angiotensin II (Ang II) induces oxidation of proteins, important in redox signaling in the cardiovascular and renal systems. The present chapter describes immunoblotting approaches to assess irreversible protein carbonylation and protein tyrosine phosphatase (PTPs) oxidation status in the proteome of vascular smooth muscle cells (VSMC).Protein carbonylation is generally measured using the OxyBlot™ approach, whereby derivatization of protein carbonyl groups (C = O) on oxidized amino acids by dinitrophenylhydrazine (DNPH) results in the formation of a stable dinitrophenyl (DNP) hydrazone product. The samples are analyzed by SDS-PAGE and a primary antibody raised against the DNP moiety is used to determine levels of irreversible protein carbonylation in the sample by immunoblotting.Oxidation of PTPs can be evaluated using a monoclonal antibody against the "hyperoxidized" (SO3H) catalytic site of these enzymes. The described methodology offers the ability to discriminate between irreversible (SO3H) and reversible (SOH) PTP oxidation states. Initially, the free unmodified PTP-thiols (S-) are alkylated and the sample is split into two. One part is used to assess the PTP-SO3H form. In the other part reversibly modified PTP-thiols are first reduced and then hyperoxidized by pervanadate (PV). Both untreated and PV-treated samples are analyzed by SDS-PAGE and "hyperoxidized" PTPs are detected by immunoblotting. The proportion of reversibly oxidized PTP-SOH fraction is determined by the difference between the signals in untreated and the PV-treated samples.The above immunoassays provide general approaches to detect and quantify global levels of irreversible protein oxidation and of irreversibly/reversibly oxidized PTPs in any (patho)physiological context. Characterization of the global redox status is essential to better understand the redox-sensitive mechanisms underlying chronic diseases associated with oxidative stress. This is particularly important in systems influenced by the renin angiotensin system, because Ang II is a potent inducer of oxidative stress and redox signaling.