Departamento de Suelos y Recursos Naturales, Facultad de Agronomía, Universidad de Concepción, Chillán, Chile; Departamento de Análisis Instrumental, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile.
Departamento de Agroindustrias, Facultad de Ingeniería Agrícola, Universidad de Concepción, Chillán, Chile.
J Chromatogr A. 2020 Jan 25;1611:460577. doi: 10.1016/j.chroma.2019.460577. Epub 2019 Sep 27.
Ultraviolet radiation from sunlight causes DNA damage in skin cells by formation of photoproducts, mainly cyclobutane pyrimidine dimers (CPD), which are reverted by exogenous CPD-photolyase, preventing photoaging and skin cancer. High performance liquid chromatography tandem mass spectrometry method for quantification of CPD-photolyase activity was developed to search new enzymes sources for dermatology or clinical studies. The method was based in the enzymatic conversion of a 15mer oligonucleotide, containing a center cyclobutane thymidine dimer, to the restored 15mer oligonucleotide. Three ion pair reagent were evaluated by response surface methodology to increase mass intensities. Additionally, chromatographic separation of oligonucleotides was performed. The selected mobile phase was 15 mM diisopropylethylamine/20 mM hexafluoroisopropanol in methanol. The method allowed total separation between the oligonucleotides studied (resolution of 2.3) by using the core shell technology, which reduce the diffusion time of the analyte into the column, increasing the efficiency and minimizing the analysis time at 7 min. The mass spectrometry detection allowed a high selectivity and sensitivity. This is the first time where MRM modality has been employed with this specific purpose. Oligonucleotides recovery from reaction mixture was ∼ 94% and the limit of quantification was 13.4 nM for 15mer. The method was evaluated with a recombinant CPD-photolyase from Synechococcus leopoliensis using purified and crude protein extract. CPD-photolyase could be measured in terms of activity for enzymatic kinetics studies, for evaluation of UV-R effects in (micro)organisms and to identify new enzymes.
阳光中的紫外线通过形成光产物（主要是环丁烷嘧啶二聚体，CPD）导致皮肤细胞中的 DNA 损伤，这些光产物可被外源性 CPD 光解酶逆转，从而防止光老化和皮肤癌。为了寻找皮肤科或临床研究的新酶源，开发了一种用于定量测定 CPD 光解酶活性的高效液相色谱串联质谱法。该方法基于含有中心环丁烷胸腺嘧啶二聚体的 15mer 寡核苷酸在酶促作用下转化为恢复的 15mer 寡核苷酸。通过响应面法评估了三种离子对试剂以增加质谱强度。此外，还对寡核苷酸进行了色谱分离。选择的流动相为 15mM 二异丙基乙胺/20mM 全氟异丙醇甲醇。该方法使用核壳技术允许研究的寡核苷酸完全分离（分辨率为 2.3），该技术减少了分析物进入色谱柱的扩散时间，从而提高了效率并将分析时间最小化至 7 分钟。质谱检测具有高选择性和灵敏度。这是首次将 MRM 模式用于此特定目的。从反应混合物中回收的寡核苷酸约为 94%，15mer 的定量限为 13.4nM。该方法使用纯化和粗蛋白提取物从 Synechococcus leopoliensis 中的重组 CPD 光解酶进行了评估。CPD 光解酶可以用于酶动力学研究的活性测定、评估（微生物）中的 UV-R 效应以及鉴定新的酶。
