Selective Affinity Enrichment of Nitrotyrosine-Containing Peptides for Quantitative Analysis in Complex Samples

Protein
tyrosine nitration by oxidative and nitrate stress is important
in the pathogenesis of many inflammatory or aging-related diseases.
Mass spectrometry analysis of protein nitrotyrosine is very challenging
because the non-nitrated peptides suppress the signals of the low-abundance
nitrotyrosine (NT) peptides. No validated methods for enrichment of
NT-peptides are currently available. Here we report an immunoaffinity
enrichment of NT-peptides for proteomics analysis. The effectiveness
of this approach was evaluated using nitrated protein standards and
whole-cell lysates in vitro. A total of 1881 NT sites were identified
from a nitrated whole-cell extract, indicating that this immunoaffinity-MS
method is a valid approach for the enrichment of NT-peptides, and
provides a significant advance for characterizing the nitrotyrosine
proteome. We noted that this method had higher affinity to peptides
with N-terminal nitrotyrosine relative to peptides with other nitrotyrosine
locations, which raises the need for future study to develop a pan-specific
nitrotyrosine antibody for unbiased, proteome-wide analysis of tyrosine
nitration. We applied this method to quantify the changes in protein
tyrosine nitration in mouse lungs after intranasal poly­(I:C) treatment
and quantified 237 NT sites. This result indicates that the immunoaffinity-MS
method can be used for quantitative analysis of protein nitrotyrosines
in complex samples.

蛋白质酪氨酸硝化（Protein tyrosine nitration）由氧化应激与硝酸盐应激介导，在多种炎症性或衰老相关疾病的发病机制中具有重要意义。质谱分析法（Mass spectrometry）用于蛋白质硝基酪氨酸的分析极具挑战性，原因在于非硝化肽会抑制低丰度硝基酪氨酸（NT）肽的信号。目前尚无经过验证的硝基酪氨酸肽富集方法。本研究报道了一种可用于蛋白质组学分析的硝基酪氨酸肽免疫亲和富集（immunoaffinity enrichment）方法。我们通过体外硝化蛋白标准品与全细胞裂解物评估了该方法的有效性。从硝化全细胞提取物中总共鉴定出1881个硝基化位点，表明该免疫亲和-质谱法是一种可行的硝基酪氨酸肽富集策略，为硝基化蛋白质组的表征研究提供了重要进展。我们注意到，相较于其他位置的硝基酪氨酸肽，该方法对N端硝基酪氨酸肽具有更高的亲和力，这提示未来需要开发泛特异性硝基酪氨酸抗体，以实现无偏倚的全蛋白质组酪氨酸硝化分析。我们将该方法应用于鼻内poly(I:C)处理后小鼠肺部的蛋白质酪氨酸硝化水平变化的定量分析，共定量到237个硝基化位点。该结果表明，免疫亲和-质谱法可用于复杂样本中蛋白质硝基酪氨酸的定量分析。