Identification and quantification of DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1) in human cells by liquid chromatography/isotope-dilution tandem mass spectrometry.
Identification and quantification of DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1) in human cells by liquid chromatography/isotope-dilution tandem mass spectrometry.
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DOI：
10.1371/journal.pone.0069894
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Dizdaroglu M
中科院分区：
文献类型：
--
作者：
Kirkali G;Jaruga P;Reddy PT;Tona A;Nelson BC;Li M;Wilson DM 3rd;Dizdaroglu M
除非修复，否则DNA损伤会导致突变或细胞死亡。因此，DNA修复蛋白可用作疾病病因学或治疗反应预测中的生物标志物。因此，DNA修复蛋白表达和基因型的准确测定是至关重要的。在参与碱基切除修复的DNA修复蛋白中，脱嘌呤/脱嘧啶核酸内切酶1（apurinic/apyrimidinic endonuclease 1，APE 1）是哺乳动物中主要的核酸内切酶，在转录调控和应激反应中起重要作用。在这里，我们提出了一种新的方法，涉及LC-MS/MS与同位素稀释，积极识别和准确定量APE 1在人类细胞和小鼠组织。制备完全15 N标记的全长人APE 1并用作内标。经过胰蛋白酶消化后，鉴定了人APE 1（hAPE 1）和15 N标记的hAPE 1的14种胰蛋白酶肽。这些肽与胰蛋白酶消化预期的理论肽相匹配，并提供了可明确鉴定hAPE 1的统计学显著性蛋白质评分。使用开发的方法，APE 1被积极确定和定量的多个人细胞系和小鼠肝脏的细胞核和细胞质提取物，使用选择性反应监测典型的质量转换的胰蛋白酶肽。我们还表明，该方法可以应用于识别在人群中发现的hAPE 1变体。这些结果描述了一种新的方法，用于准确测量野生型和变异形式的hAPE 1在体内，并最终确定这种蛋白质在疾病发展和治疗反应中的作用。
Unless repaired, DNA damage can drive mutagenesis or cell death. DNA repair proteins may therefore be used as biomarkers in disease etiology or therapeutic response prediction. Thus, the accurate determination of DNA repair protein expression and genotype is of fundamental importance. Among DNA repair proteins involved in base excision repair, apurinic/apyrimidinic endonuclease 1 (APE1) is the major endonuclease in mammals and plays important roles in transcriptional regulation and modulating stress responses. Here, we present a novel approach involving LC-MS/MS with isotope-dilution to positively identify and accurately quantify APE1 in human cells and mouse tissue. A completely 15N-labeled full-length human APE1 was produced and used as an internal standard. Fourteen tryptic peptides of both human APE1 (hAPE1) and 15N-labeled hAPE1 were identified following trypsin digestion. These peptides matched the theoretical peptides expected from trypsin digestion and provided a statistically significant protein score that would unequivocally identify hAPE1. Using the developed methodology, APE1 was positively identified and quantified in nuclear and cytoplasmic extracts of multiple human cell lines and mouse liver using selected-reaction monitoring of typical mass transitions of the tryptic peptides. We also show that the methodology can be applied to the identification of hAPE1 variants found in the human population. The results describe a novel approach for the accurate measurement of wild-type and variant forms of hAPE1 in vivo, and ultimately for defining the role of this protein in disease development and treatment responses.
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影响因子：
9.9
作者：
Lange, Vinzenz;Picotti, Paola;Domon, Bruno;Aebersold, Ruedi
通讯作者：
Aebersold, Ruedi
影响因子：
3.8
作者：
Demple, B;Sung, JS
通讯作者：
Sung, JS
DOI：
10.1073/pnas.88.24.11450
发表时间：
1991-12-01
影响因子：
11.1
作者：
DEMPLE, B;HERMAN, T;CHEN, DS
通讯作者：
CHEN, DS
影响因子：
9.9
作者：
Hayward, C;Colville, S;Brock, DJH
通讯作者：
Brock, DJH
影响因子：
2.9
作者：
BRADFORD, MM
通讯作者：
BRADFORD, MM