Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA.
J Proteome Res. 2013 Feb 1;12(2):1049-61. doi: 10.1021/pr301037t. Epub 2013 Jan 11.
Accumulated evidence points to DNA repair capacity as an important factor in cancer and other diseases. DNA repair proteins are promising drug targets and are emerging as prognostic and therapeutic biomarkers. Thus, the knowledge of the overexpression or underexpression levels of DNA repair proteins in tissues will be of fundamental importance. In this work, mass spectrometric assays were developed for the measurement in tissues of the human DNA repair protein NEIL1 (hNEIL1), which is involved in base excision and nucleotide excision repair pathways of oxidatively induced DNA damage. Liquid chromatography/isotope-dilution tandem mass spectrometry (LC-MS/MS), in combination with a purified and fully characterized recombinant (15)N-labeled analogue of hNEIL1 ((15)N-hNEIL1) as an internal standard, was utilized to develop an accurate method for the quantification of hNEIL1. Both hNEIL1 and (15)N-hNEIL1 were hydrolyzed with trypsin, and 18 tryptic peptides from each protein were identified by LC-MS/MS on the basis of their full-scan mass spectra. These peptides matched the theoretical peptides expected from trypsin hydrolysis of hNEIL1 and provided a statistically significant protein score that would unequivocally identify hNEIL1. The product ion spectra of the tryptic peptides from both proteins were recorded, and the characteristic product ions were defined. Selected-reaction monitoring was used to analyze mixtures of hNEIL1 and (15)N-hNEIL1 on the basis of product ions. Additional confirmation of positive identification was demonstrated via separation of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in-gel tryptic digestion followed by LC-MS/MS analysis. These results suggest that the developed assays would be highly suitable for the in vivo positive identification and accurate quantification of hNEIL1 in tissues.
越来越多的证据表明，DNA 修复能力是癌症和其他疾病的一个重要因素。DNA 修复蛋白是很有前途的药物靶点，并且正在成为预后和治疗的生物标志物。因此，了解组织中 DNA 修复蛋白的过度表达或低表达水平将是至关重要的。在这项工作中，开发了用于测量人体 DNA 修复蛋白 NEIL1（hNEIL1）在组织中的表达水平的质谱检测方法，该蛋白参与氧化诱导的 DNA 损伤的碱基切除和核苷酸切除修复途径。液相色谱/同位素稀释串联质谱（LC-MS/MS）与纯化和完全表征的重组（15）N 标记的 hNEIL1 类似物（（15）N-hNEIL1）作为内标结合使用，开发了一种准确测量 hNEIL1 的方法。用胰蛋白酶水解 hNEIL1 和（15）N-hNEIL1，根据它们的全扫描质谱鉴定出两种蛋白质的 18 种胰酶肽。这些肽与预期来自 hNEIL1 胰酶水解的理论肽相匹配，并提供了具有统计学意义的蛋白质得分，可明确识别 hNEIL1。记录了两种蛋白质的胰酶肽的产物离子谱，并定义了特征产物离子。基于产物离子，使用选择反应监测分析 hNEIL1 和（15）N-hNEIL1 的混合物。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离蛋白质并进行胶内胰酶消化，然后进行 LC-MS/MS 分析，进一步证实了阳性鉴定。这些结果表明，所开发的检测方法非常适合在体内对 hNEIL1 进行阳性鉴定和准确定量。
