用于研究蛋白质 S-亚硝基化的位点特异性蛋白质组学方法。

Site-specific proteomics approach for study protein S-nitrosylation.

机构信息

Department of Pathology and Microbiology, Mass Spectrometry and Proteomics Core Facility, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.

出版信息

Anal Chem. 2010 Sep 1;82(17):7160-8. doi: 10.1021/ac100569d.

DOI:10.1021/ac100569d

PMID:20687582

Abstract

Here we present a novel and robust method for the identification of protein S-nitrosylation sites in complex protein mixtures. The approach utilizes the cysteinyl affinity resin to selectively enrich S-nitrosylated peptides reduced by ascorbate followed by nanoscale liquid chromatography tandem mass spectrometry. Two alkylation agents with different added masses were employed to differentiate the S-nitrosylation sites from the non-S-nitrosylation sites. We applied this approach to MDA-MB-231 cells treated with Angeli's salt, a nitric oxide donor that has been shown to inhibit breast tumor growth and angiogenesis. A total of 162 S-nitrosylation sites were identified and an S-nitrosylation motif was revealed in our study. The 162 sites are significantly more than the number reported by previous methods, demonstrating the efficiency of our approach. Our approach will further facilitate the functional study of protein S-nitrosylation in cellular processes and may reveal new therapeutic targets.

摘要

我们在此提出了一种新颖而强大的方法，用于鉴定复杂蛋白质混合物中的蛋白质 S-亚硝酰化位点。该方法利用半胱氨酸亲和树脂选择性地富集被抗坏血酸还原的 S-亚硝酰化肽，然后进行纳升级液相色谱串联质谱分析。我们使用了两种不同添加质量的烷基化试剂来区分 S-亚硝酰化位点和非 S-亚硝酰化位点。我们将这种方法应用于用 Angeli's salt 处理的 MDA-MB-231 细胞，Angeli's salt 是一种一氧化氮供体，已被证明可以抑制乳腺癌生长和血管生成。我们共鉴定到 162 个 S-亚硝酰化位点，并在我们的研究中揭示了一个 S-亚硝酰化模体。与以前的方法相比，这 162 个位点明显更多，证明了我们方法的效率。我们的方法将进一步促进细胞过程中蛋白质 S-亚硝酰化的功能研究，并可能揭示新的治疗靶点。