高效纯化和基于 LC ms/ms 的十-十一 Translocation-2 5-甲基胞嘧啶酶的检测开发
Summary
在这里, 我们提出了一个有效的单步纯化活性无标签人的协议十-十一 translocation-2 (TET2) 5-甲基胞嘧啶酶使用离子交换色谱法及其测定液相色谱-串联质量基于质谱 (LC-ms) 的方法。
Abstract
5-甲基胞嘧啶 (5mC) 介导的表观遗传转录调控在真核发育中起着至关重要的作用。这些表观遗传标记的去甲基化是通过顺序氧化十-十一易位芳香烃 (TET1-3), 其次是胸腺嘧啶 DNA glycosylase 依赖基础切除修复。由于基因突变或其他表观遗传机制, TET2 基因的失活与不同癌症, 特别是造血恶性肿瘤患者预后较差有关。在这里, 我们描述了一个有效的单步纯化酶活性无标签人 TET2 酶使用阳离子交换层析。我们进一步提供液相色谱-串联质谱 (LC ms/ms) 方法, 可以分离和量化四个正常的 DNA 基础 (a, T, G 和 C), 以及四个改性胞嘧啶碱基 (5-甲基, 5-羟甲基, 5-甲酰基, 和 5-羧基)。本试验可用于评价野生型和突变 TET2 芳香烃的活性。
Introduction
在 CpG 二核苷酸内胞嘧啶碱基的 C5 位置是哺乳动物基因组1中主要的甲基化部位 (5mCpG)。此外, 一些最近的研究发现在非 CpG 站点 (5mCpH, 其中 H = a, T, 或 C)2,3的广泛 C5 胞嘧啶甲基化 (5mC)。5mC 修饰在内源座子和基因促进剂3、4、5中用作转录消声器。5mC DNA 甲基化在 X 染色体失活、基因印迹、核重编程和组织特异基因表达5、6、7等方面也起着重要作用。C5 位置的胞嘧啶甲基化是由 DNA 甲基转移酶进行的, 而这些酶的突变导致重大发育缺陷8。5mC 标记的去除由 TET1-3 5mC 氧化酶9、10启动。这些春节家庭芳香烃通过连续氧化步骤11,12,13转换5mC 到 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) 和 5-carboxylcytosine (5caC)。最后, 胸腺嘧啶 DNA glycosylase 替代5fC 或5caC 到未修改的胞嘧啶使用基础切除修复途径11。
人类TET2基因被确定为在不同的造血恶性肿瘤, 包括骨髓增生异常综合征 (mds)14,15,16, mds-骨髓增生性肿瘤经常突变的基因 (mds-mpn) 和急性髓系白血病 (AML) 源自 mds 和 mds-MPN16。与野生型 (wt) TET214相比, TET2 突变患者骨髓 DNA 的5hmC 修饰水平较低。一些小组已经开发出 TET2-knockout 小鼠模型, 以阐明其在正常造血和髓样转化中的作用17,18,19,20。这些在 TET2 基因突变的小鼠最初是正常和可行的, 但表现出不同的造血恶性肿瘤, 因为他们的年龄导致他们的早期死亡。这些研究表明 wt-TET2 在正常造血分化中发挥的重要作用。在这些小鼠模型中, 杂合造血干细胞 (TET2+/造血干细胞) 和纯合 TET2造血干细胞在 wt-TET2 造血谱系中具有优于纯合造血干细胞重新填充的竞争优势, 同时 TET2TET2 造血干细胞开发了多种造血恶性肿瘤17,18。这些研究表明, TET2 酶的 haploinsufficiency 改变了造血干细胞的发展, 导致了造血恶性肿瘤。
与 TET2 基因突变的小鼠相似, 大多数白血病患者 haploinsufficiency TET2 酶活动的表现。这些主要杂合体细胞突变包括帧移位和废话突变分散在整个 TET2 基因体内, 而错义突变是最聚集在酶领域12。到目前为止, 文献中很少有关于 wt 和 mutant-TET2 的表征, 主要是由于 TET2 酶的生产和测定21的困难。在这里, 我们报告了一个简单的单步纯化原生 TET2 酶使用离子交换色谱法。进一步, 对 TET2 酶的酶活性进行了优化, 并进行了定量的 LC-ms/ms 测定。
Protocol
Representative Results
Discussion
TET2 基因突变是在不同的造血恶性肿瘤患者中发现的最常见的遗传变化。到目前为止, 数以百计的不同的 TET2 突变, 其中包括废话, 帧移位, 和错义突变, 已确定在患者12。与 wt-TET214相比, TET2突变的患者在骨髓中的基因组5hmC 水平低。突变TET2实验概括了这些突变对转染细胞中5hmC 水平的影响14。结果 TET2-knockout 小鼠模型表明, TET2 酶水?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究由美国国防部资助的形式的想法奖 (W81XWH-13-1-0174), 再生障碍性贫血 & MDS 基金会赠款, 和 UMRB 授予 M.M. 作者感谢莫希特贾斯瓦尔和 Subhradeep 巴尔在 TET2 向量的初始克隆 pDEST14。
Materials
| HEPES | Carbosynth | FH31182 | |
| Iron(II) sulfate heptahydrate | Sigma-Aldrich | F8633 | |
| α-Ketoglutaric acid (2-Oxoglutaric acid) | Sigma-Aldrich | K1750 | |
| L-Ascorbic acid | Sigma-Aldrich | A4544 | |
| Ethylenediaminetetraacetic acid disodium salt dihydrate | Sigma-Aldrich | E5134 | |
| Ammonium acetate | Sigma-Aldrich | A1542 | |
| Acetonitrile | Fisher Scientific | 75-05-8 | |
| HPLC grade water | Fisher Scientific | 7732-18-5 | |
| Oligo clean and concentrator | Zymo Research | D4061 | |
| DNAse I | New England Biolabs | M0303S | |
| S1 Nuclease | Thermo Scientific | ENO321 | |
| CIAP (Calf intestinal alkaline phosphatase) | New England Biolabs | M0290S | |
| LB Media | Affymetrix | J75852 | |
| IPTG | Carbosynth | EI05931 | |
| MES [2-(N-Morpholino)ethanesulfonic acid monohydrate] | Carbosynth | FM37015 | |
| Sodium chloride | Fisher Scientific | 7647-14-5 | |
| Glycerol | Sigma-Aldrich | G7893 | |
| SP Sepharose | Fisher Scientific | 45-002-934 | |
| 2'-Deoxy-5-methylcytidine | TCI | D3610 | |
| 2'-Deoxy-5-hydroxymethyalcytidine | TCI | D4220 | |
| 2'-Deoxycytidine-5-carboxylic acid, sodium salt | Berry & Associates | PY 7593 | |
| 5-Formyl-2'-deoxycytidine | Berry & Associates | PY 7589 | |
| 2'-Deoxycytidine | Berry & Associates | PY 7216 | |
| 2'-Deoxyadenosine | Carbosynth | ND04011 | |
| 2'-Deoxyguanosine | Carbosynth | ND06306 | |
| 2'-Deoxythymidine | VWR Life Science | 97061-764 | |
| Gateway technology | Thermo Fisher | 11801016 | |
| Beckman Allegra X-15R centrifuge | Beckman Coulter | 392932 | |
| Sonic Dismembrator 550 | Fisher Scientific | XL2020 | |
| ÄKTA FPLC system | Pharmacia (GE Healthcare) | 18116468 | |
| FreeZone 4.5 freeze dry system | Labconco | 7750020 | |
| Zymo Oligo purification columns | Zymo Research | D4061 | |
| BDS Hypersil C18 column | Keystone Scientific, INC | 105-46-3 | |
| 3200 Q-Trap mass spectrometer | AB Sciex | ||
| HPLC | Shimadzu HPLC | ||
| XK16/20 FPLC column | Pharmacia (GE Healthcare) | 28988937 |
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