ANSID: A Solid-Phase Proteomic Approach for Identification and Relative Quantification of Aromatic Nitration Sites.
ANSID: A Solid-Phase Proteomic Approach for Identification and Relative Quantification of Aromatic Nitration Sites.
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DOI：
10.3389/fchem.2015.00070
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发表时间：
2015
影响因子：
5.5
通讯作者：
Gross SS
中科院分区：
文献类型：
--
作者：
Nuriel T;Whitehouse J;Ma Y;Mercer EJ;Brown N;Gross SS
蛋白质中酪氨酸和其他芳香族氨基酸残基的硝化作用发生在炎症、神经退行性疾病和心血管疾病的背景下，重要的是，这种修饰与多种疾病的发病机制和衰老的生理过程有关。为了了解芳香族硝化在健康和疾病中的生物学后果，从分子上鉴定经历硝化的蛋白质，指定其同源修饰位点并量化其硝化程度至关重要。迄今为止，硝化蛋白质的无偏鉴定通常涉及艰苦的2D凝胶电泳，然后用抗硝基酪氨酸抗体进行Western印迹检测。除了相对缓慢和费力之外，该方法还具有覆盖范围有限、假阳性鉴定的可能性以及不能揭示特定氨基酸修饰位点的缺点。为了克服这些缺点，我们开发了一种固相化学捕获方法，用于蛋白质中硝基酪氨酸和硝基色氨酸位点的无偏和高通量发现。利用这种方法，我们已经成功地确定了几个内源性硝化蛋白质在大鼠大脑和总共244硝化肽从145蛋白质在体外暴露的大鼠大脑匀浆的硝化剂过氧亚硝酸盐（1 mM）。正如预期的那样，Tyr残基构成了绝大多数过氧亚硝酸盐介导的蛋白质硝化位点;然而，我们惊讶地发现了几种含有硝化Trp残基的脑蛋白。通过引入稳定同位素标记步骤，这种新的芳香族硝化位点鉴定（ANSID）方法也适用于蛋白质中硝化位点丰度的相对定量。ANSID方法的应用为我们进一步了解蛋白质硝化在疾病发病机制和正常生理中的作用提供了巨大的潜力。
Nitration of tyrosine and other aromatic amino acid residues in proteins occurs in the setting of inflammatory, neurodegenerative, and cardiovascular diseases—importantly, this modification has been implicated in the pathogenesis of diverse diseases and the physiological process of aging. To understand the biological consequences of aromatic nitration in both health and disease, it is critical to molecularly identify the proteins that undergo nitration, specify their cognate modification sites and quantify their extent of nitration. To date, unbiased identification of nitrated proteins has often involved painstaking 2D-gel electrophoresis followed by Western Blotting with an anti-nitrotyrosine antibody for detection. Apart from being relatively slow and laborious, this method suffers from limited coverage, the potential for false-positive identifications, and failure to reveal specific amino acid modification sites. To overcome these shortcomings, we have developed a solid-phase, chemical-capture approach for unbiased and high-throughput discovery of nitrotyrosine and nitrotryptophan sites in proteins. Utilizing this method, we have successfully identified several endogenously nitrated proteins in rat brain and a total of 244 nitrated peptides from 145 proteins following in vitro exposure of rat brain homogenates to the nitrating agent peroxynitrite (1 mM). As expected, Tyr residues constituted the great majority of peroxynitrite-mediated protein nitration sites; however, we were surprised to discover several brain proteins that contain nitrated Trp residues. By incorporating a stable-isotope labeling step, this new Aromatic Nitration Site IDentification (ANSID) method was also adapted for relative quantification of nitration site abundances in proteins. Application of the ANSID method offers great potential to advance our understanding of the role of protein nitration in disease pathogenesis and normal physiology.