年龄相关性白内障晶状体蛋白磷酸化位点鉴定

发布时间：2018-05-29 14:11

本文选题：年龄相关性白内障 + 磷酸化　；参考：《复旦大学》2012年博士论文

【摘要】：白内障是全球首位致盲眼病,而年龄相关性白内障(age related cataract,ARC)也是老年人视力下降的主要原因。目前手术仍是治疗白内障唯一有效的方法,由于我国人口数量庞大,经济欠发达,高昂的手术费用会给社会带来沉重的经济负担。因此,预防或者推迟ARC发病,无疑具有重要意义。磷酸化是一种常见蛋白质翻译后修饰,磷酸化与去磷酸化这一可逆的过程调节着信号传导、基因表达等多种细胞生物学进程,而蛋白质磷酸化修饰的异常会引发一系列疾病。晶状体蛋白是人晶状体内含量最高的结构与功能蛋白,研究者推测晶状体蛋白的磷酸化与去磷酸化的过程失调可能是ARC发病的早期改变或者说是晶状体蛋白变性前的病变。由于晶状体蛋白磷酸化过程是可逆的,因此在晶状体蛋白尚未发生变性的早期,也即白内障形成之前是干预ARC发病的有效时机。鉴定晶状体蛋白的磷酸化位点,将是进一步探索位点功能的第一步。本实验首次利用原位酶解联合基质辅助激光解析电离质谱鉴定人透明品状体与ARC晶状体组织切片中的磷酸化位点。研究者在人透明晶状体中鉴定到了4种晶状体蛋白中的8个磷酸化位点；在ARC晶状体中鉴定到了6种晶状体蛋白中的11个磷酸化位点,其中9个磷酸化位点先前未有报道。本实验为进一步的磷酸化位点质谱成像定位和磷酸化位点的功能学研究奠定了基础。目的：探求人晶状体组织冰冻切片的最佳条件并优化基质辅助激光解析电离质谱对晶状体组织切片鉴定的实验参数。方法：(1)将-80℃冻存的人透明晶状体与ARC晶状体分别在-10℃至-25℃温度下沿赤道部轴向切片,切片厚度10μm至20μm,用解剖镜观察所得切片,确定最适宜的温度与切片厚度。 (2)使用标准蛋白细胞色素C与马心肌红蛋白作为标准蛋白,从基质种类、基质喷涂层数、喷涂速度等各方面优化并探寻最佳的基质辅助激光解析电离质谱鉴定晶状体组织切片中磷酸化位点的实验参数。结果：经过解剖镜观察,最适宜的切片温度为-18℃,切片厚度为141μm。此条件下切片完整性好,易于转移和进一步的质谱鉴定。基质辅助激光解析电离质谱鉴定人晶状体组织切片的最佳实验条件为：以加入0.1%三氟乙酸、0.6-0.8mg/ml柠檬酸氨的浓度为2.5mg/mL的α-氰基-4-羟基-肉桂酸(50%乙腈：50%水)为基质,直接喷涂在组织切片表面；加热温度120℃,基质流速O.100ml/min,喷头移动速度200mm/min,晾干后进行质谱检测。所得质谱数据使用DataExPlorer4.5进行后期处理。结论：确立了人晶状体组织冰冻切片的最佳条件,并优化了基质辅助激光解析电离质谱对人晶状体组织切片鉴定的实验条件与实验参数,为进一步的研究奠定了基础。目的：在优化了的实验条件下,利用原位酶解联合基质辅助激光解析电离质谱鉴定年龄相关性白内障晶状体及人透明晶状体中晶状体蛋白的磷酸化位点并比较其差异。方法：(1)将-80℃冻存的人透明晶状体与年龄相关性白内障晶状体在-18℃温度下沿赤道部轴向切片,切片厚度14μm,随后将组织切片转移至盖玻片上。 (2)用肌红蛋白、牛血清白蛋和α-酪蛋白作为标准蛋白对胰蛋白酶-石墨烯固定化酶反应器的酶解效率进行考察。 (3)利用胰蛋白酶-石墨烯固定化酶反应器联合基质辅助激光解析电离质谱鉴定人透明晶状体与年龄相关性白内障晶状体组织切片中的磷酸化位点,所得质谱数据使用DataExPlorer4.5进行后期处理。结果：研究者在人透明晶状体中鉴定到了4种晶状体蛋白中的8个磷酸化位点；在年龄相关性白内障晶状体中鉴定到了6种晶状体蛋白中的11个磷酸化位点,其中9个磷酸化位点先前未有报道。结论：首次利用原位酶解联合基质辅助激光解析电离质谱在年龄相关性白内障品状体中鉴定到了6种晶状体蛋白中的11个磷酸化位点,为进一步的质谱成像定位和磷酸化位点的功能性研究奠定了基础。
[Abstract]:Cataract is the leading cause of blindness in the world, and age related cataract (ARC) is also the main reason for the decline of visual acuity in the elderly. Surgery is still the only effective method for the treatment of cataract. Because of the huge population, less developed economy and high cost of operation, it will bring a heavy economic burden to the society. It is of great importance to prevent or delay the onset of ARC.
Phosphorylation is a common post-translational modification of protein. The reversible process of phosphorylation and dephosphorylation regulates a variety of cellular biological processes, such as signal transduction, gene expression, and so on, and the abnormal phosphorylation of proteins leads to a series of diseases. The crystalline protein is the highest content structure and functional protein in the human crystalline body. It is speculated that the imbalance of phosphorylation and dephosphorylation of lens protein may be an early change in ARC pathogenesis or a pathological change before lens protein degeneration.
Since the phosphorylation of the crystallin is reversible, it is an effective time to intervene in the pathogenesis of ARC before the lens protein has not been denatured. The identification of the phosphorylation site of the lens protein will be the first step to further explore the function of the loci.
This experiment was the first time to identify phosphorylation sites in human transparent substance and ARC lens tissue slices by in situ enzyme solution combined with matrix assisted laser analytical ionization mass spectrometry. The researchers identified 8 phosphorylation sites in 4 kinds of lens proteins in the human transparent lens; 11 of the 6 kinds of lens proteins were identified in the ARC lens. A phosphorylation site, of which 9 phosphorylation sites have not been reported previously.
This study laid a foundation for further localization of phosphorylation site mass spectrometry and functional study of phosphorylation sites.
Objective: To explore the optimum conditions for frozen section of human lens and optimize the experimental parameters of the identification of lens tissue by matrix assisted laser analytical ionization mass spectrometry.
Methods: (1) the transparent lens and ARC lens, frozen at -80 C, were sliced along the equator at the temperature of -10 and -25, respectively. The thickness of the slice was 10 m to 20 u m, and the sections were observed with the anatomical mirror to determine the optimum temperature and thickness of the slice.
(2) using the standard protein cytochrome C and the horse heart myoglobin as the standard protein, the test parameters for the identification of phosphorylation sites in the lens tissue sections are optimized from the types of matrix, the number of stroma spraying layers, and the speed of spraying.
Results: the most suitable slice temperature was -18 C, and the thickness of the slice was 141 u m.. The section integrity was good, the transfer and mass spectrometric identification were easy under this condition. The best experimental condition for the identification of human lens tissue section by matrix assisted laser analytical ionization mass spectrometry was to add 0.1% three FLUOROACETIC acid and 0.6-0.8mg/ml citric acid. The concentration of ammonia is 2.5mg/mL cyanohydroxyl -4- hydroxyl cinnamic acid (50% acetonitrile: 50% water) as matrix and directly sprayed on the surface of tissue section; the heating temperature is 120 C, the velocity of matrix is O.100ml/min, the velocity of the nozzle is 200mm/min, and the mass spectrometry after drying is carried out. The mass mass data is treated by DataExPlorer4.5 for late treatment.
Conclusion: the optimum conditions for freezing section of human lens tissue were established, and the experimental conditions and experimental parameters for the identification of human lens tissue section by matrix assisted laser analytical ionization mass spectrometry were optimized, which laid a foundation for further research.
Objective: to identify the phosphorylation sites of the age-related cataract lens and the human transparent lens MICROTEK body protein by in situ enzyme solution combined with matrix assisted laser analytical ionization mass spectrometry and compare the difference between them.
Methods: (1) the transparent lens stored at -80 C and the age-related cataract lens were sliced along the equator at the temperature of -18 C at the temperature of -18 C, and the thickness of the slice was 14 m, then the tissue sections were transferred to the cover glass.
(2) using myoglobin, bovine blood albumin and alpha casein as the standard protein, the enzymatic efficiency of trypsin graphene immobilized enzyme reactor was investigated.
(3) using the trypsin graphene immobilized enzyme reactor and the matrix assisted laser analytical ionization mass spectrometry to identify the phosphorylation sites in the transparent lens and the age-related cataract lens tissue sections. The mass spectrum data were processed by DataExPlorer4.5 for later treatment.
Results: the researchers identified 8 phosphorylation sites in the 4 kinds of lens proteins in the human transparent lens; 11 phosphorylation sites of 6 kinds of lens proteins were identified in the age-related cataract lens, of which 9 phosphorylation sites were not previously reported.
Conclusion: for the first time, 11 phosphorylation sites in 6 kinds of lens proteins were identified by in situ enzymolysis combined with matrix assisted laser analytical ionization mass spectrometry in age related cataract bodies, which laid the foundation for further functional study of mass spectrometry location and phosphorylation sites.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R776.1

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