Steinbeis Research and Transfer Center for Biopolymer Analysis, Department of Chemistry, University of Konstanz, Konstanz, Germany.
J Am Soc Mass Spectrom. 2012 Nov;23(11):1831-40. doi: 10.1007/s13361-012-0461-4. Epub 2012 Aug 21.
Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer's disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar K(D) values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.
蛋白质中的酪氨酸硝化在生理条件下发生，并在与氧化应激相关的疾病条件下增加，如炎症和阿尔茨海默病。鉴定和量化酪氨酸硝化对于理解硝化机制及其功能后果至关重要。质谱（MS）最适合鉴定硝化位点，但受到硝化诱导的低稳定性和修饰水平以及可能的结构变化的限制。在这一见解中，我们讨论了使用硝基酪氨酸（NT）特异性抗体通过蛋白水解亲和提取来鉴定和量化硝化位点的方法，结合电喷雾-MS。这种方法的效率通过鉴定来自几个生物样本的嗜酸性粒细胞颗粒中的两种蛋白质（嗜酸性粒细胞阳离子蛋白（ECP）和嗜酸性粒细胞衍生的神经毒素（EDN）中的特定硝化位点来说明。亲和提取与 Edman 测序相结合，使硝化水平的定量成为可能，ECP 和 EDN 的硝化水平分别为 8%和 15%。利用可用晶体结构进行结构建模和使用合成 NT-肽进行的亲和研究表明，酪氨酸硝化序列基序包括 NT-残基附近带正电荷的残基，位于蛋白质结构的特定表面可及位点。通过在线生物亲和-MS 测定的 ECP 和 EDN 的 Tyr-硝化肽与 NT 抗体的亲和力提供了纳摩尔 K(D) 值。相比之下，在使用 NT 特异性抗体时，从囊性纤维化患者的蛋白质中获得了酪氨酸硝化的假阳性鉴定，并且被证明是羟基酪氨酸修饰。这些结果表明，亲和-MS 方法对于明确鉴定生物学酪氨酸硝化至关重要。
