年龄相关性白内障晶状体蛋白磷酸化位点鉴定
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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。
Cataract is the main cause of vision impairment in the world, and age-related cataract (ARC) is the most common reason of blindness in elder people. To date, surgery is still the only way to cure cataract, but the high cost has become a tremendous burden for our society. Therefore, preventing or delaying ARC is of great significance.
Protein phosphorylation, one of the most important protein post translational modifications, plays a key role in regulating many cellular processes such as proliferation, differentiation, and signal transduction. However, the abnormality of this reversible phosphorylation in proteins may result in a wide variety of diseases. As the major protein components of the vertebrate eye lens, crystallins have to last the life time and retain lens transparence. For the aforementioned reasons, we believe that the imbalanced protein phosphorylation of crystallins may result in the early change of lens before the irreversible crystallin denaturation. Therefore, preventing cataract before the irreversible protein denaturation is considered to be the optimum option. In addition, identification of phosphorylation sites in crystallins is the first step of functional study.
In this study, we used in situ tryptic digestion combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify and compare the phosphorylation sites in crystallins of a human transparent lens and an age related cataract lens. In the transparent lens tissue, four crystallins were identified to contain a total of eight sites of phosphorylation. While in the ARC lens, six crystallins were found to contain eleven sites of phosphorylation, nine of which were previously unreported.
In summary, a method for in situ tryptic digestion combined with MALDI tissue identification of crystallin phosphorylation products has been developed. The observations of phosphorylation sites will be useful in spatial distribution imaging and functional study.
Methods:(1) Frozen human lenses were equatorially and axially cryosectioned into10μm to20μm thick sections under the temperature of-10℃to-25℃respectively, and the sections were mounted onto glass slides. Dissecting microscope was used to determine the optimum temperature and thickness of lens cryosection.
(2) Cytochrome C and myoglobin were used as standard proteins to optimize the experimental condition of MALDI mass spectrometry.
Results:The optimum cryosection temperature was-18℃and the section thickness is14μm. The optimal matrix was composed of2.5mg/mL alpha-Cyano-4-hydroxycinnamic acid (CHCA) with0.1%trifluoracetic acid (TFA) and0.6-0.8mg/ml citric acid ammonia. Two layers of matrix were sprayed on the section with the velocity of200mm/min at the temperature of120℃. MALDI mass spectra were acquired in reflection mode between m/z700and4000with a Nd:YAG laser at355nm, a repetition rate of200Hz, and an acceleration voltage of20kV. Two thousand laser shots per spot were accumulated at50different spot positions. The data was further analyzed by Data Explorer4.5.
Conclusions:We optimized the experimental condition of lens cryosection and MALDI mass spectrometry and laid the foundation for further study.
Purpose:The purpose of this part was to identify and compare the phosphorylation sites in crystallins of a human transparent lens and an age related cataract lens using in situ tryptic digestion combined with matrix-assisted laser desorption/ionization mass spectrometry.
Methods:(1) Frozen human lenses were equatorially and axially cryosectioned into14-μm-thick sections at the temperature of-18℃, and the sections were mounted onto glass slides.
(2) Myoglobin, bovine serum albumin (BSA) and a-casein were used as standard proteins to test the efficiency of Graphene-Immobilized Trypsin Reactor.
(3) Proteins in the lens sections were digested with Graphene-Immobilized Trypsin Reactor and subjected to MALDI mass spectrometric analysis for identification of modifications. The data was further analyzed by Data Explorer4.5.
Results:In the transparent lens tissue, four crystallins were identified to contain a total of eight sites of phosphorylation. While in the ARC lens, six crystallins were found to contain eleven sites of phosphorylation, nine of which were previously unreported.
Conclusions:In summary, a method for in situ tryptic digestion combined with MALDI tissue identification of crystallin products has been developed. The observations of phosphorylation sites will be useful in spatial distribution imaging and functional study.
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。
Cataract is the main cause of vision impairment in the world, and age-related cataract (ARC) is the most common reason of blindness in elder people. To date, surgery is still the only way to cure cataract, but the high cost has become a tremendous burden for our society. Therefore, preventing or delaying ARC is of great significance.
Protein phosphorylation, one of the most important protein post translational modifications, plays a key role in regulating many cellular processes such as proliferation, differentiation, and signal transduction. However, the abnormality of this reversible phosphorylation in proteins may result in a wide variety of diseases. As the major protein components of the vertebrate eye lens, crystallins have to last the life time and retain lens transparence. For the aforementioned reasons, we believe that the imbalanced protein phosphorylation of crystallins may result in the early change of lens before the irreversible crystallin denaturation. Therefore, preventing cataract before the irreversible protein denaturation is considered to be the optimum option. In addition, identification of phosphorylation sites in crystallins is the first step of functional study.
In this study, we used in situ tryptic digestion combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify and compare the phosphorylation sites in crystallins of a human transparent lens and an age related cataract lens. In the transparent lens tissue, four crystallins were identified to contain a total of eight sites of phosphorylation. While in the ARC lens, six crystallins were found to contain eleven sites of phosphorylation, nine of which were previously unreported.
In summary, a method for in situ tryptic digestion combined with MALDI tissue identification of crystallin phosphorylation products has been developed. The observations of phosphorylation sites will be useful in spatial distribution imaging and functional study.
Methods:(1) Frozen human lenses were equatorially and axially cryosectioned into10μm to20μm thick sections under the temperature of-10℃to-25℃respectively, and the sections were mounted onto glass slides. Dissecting microscope was used to determine the optimum temperature and thickness of lens cryosection.
(2) Cytochrome C and myoglobin were used as standard proteins to optimize the experimental condition of MALDI mass spectrometry.
Results:The optimum cryosection temperature was-18℃and the section thickness is14μm. The optimal matrix was composed of2.5mg/mL alpha-Cyano-4-hydroxycinnamic acid (CHCA) with0.1%trifluoracetic acid (TFA) and0.6-0.8mg/ml citric acid ammonia. Two layers of matrix were sprayed on the section with the velocity of200mm/min at the temperature of120℃. MALDI mass spectra were acquired in reflection mode between m/z700and4000with a Nd:YAG laser at355nm, a repetition rate of200Hz, and an acceleration voltage of20kV. Two thousand laser shots per spot were accumulated at50different spot positions. The data was further analyzed by Data Explorer4.5.
Conclusions:We optimized the experimental condition of lens cryosection and MALDI mass spectrometry and laid the foundation for further study.
Purpose:The purpose of this part was to identify and compare the phosphorylation sites in crystallins of a human transparent lens and an age related cataract lens using in situ tryptic digestion combined with matrix-assisted laser desorption/ionization mass spectrometry.
Methods:(1) Frozen human lenses were equatorially and axially cryosectioned into14-μm-thick sections at the temperature of-18℃, and the sections were mounted onto glass slides.
(2) Myoglobin, bovine serum albumin (BSA) and a-casein were used as standard proteins to test the efficiency of Graphene-Immobilized Trypsin Reactor.
(3) Proteins in the lens sections were digested with Graphene-Immobilized Trypsin Reactor and subjected to MALDI mass spectrometric analysis for identification of modifications. The data was further analyzed by Data Explorer4.5.
Results:In the transparent lens tissue, four crystallins were identified to contain a total of eight sites of phosphorylation. While in the ARC lens, six crystallins were found to contain eleven sites of phosphorylation, nine of which were previously unreported.
Conclusions:In summary, a method for in situ tryptic digestion combined with MALDI tissue identification of crystallin products has been developed. The observations of phosphorylation sites will be useful in spatial distribution imaging and functional study.
引文
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37. Ma Z, Hanson SR, Lampi KJ, David LL, Smith DL, Smith JB. Age-related changes in human lens crystallins identified by HPLC and mass spectrometry. Exp Eye Res1998 Jul;67(1):21-30.
38. Lampi KJ, Ma Z, Shih M, Shearer TR, Smith JB, Smith DL, David LL. Sequence analysis of betaA3, betaB3, and betaA4 crystallins completes the identification of the major proteins in young human lens. J Biol Chem 1997 Jan 24;272(4):2268-75.
39. Billingsley G, Santhiya ST, Paterson AD, Ogata K, Wodak S, Hosseini SM, Manisastry SM, Vijayalakshmi P, Gopinath PM, Graw J, Heon E. CRYBA4, a novel human cataract gene, is also involved in microphthalmia. Am J Hum Genet2006 Oct;79(4):702-9.
40. Zhou G, Zhou N, Hu S, Zhao L, Zhang C, Qi Y. A missense mutation in CRYBA4 associated with congenital cataract and microcornea. Mol Vis2010;16:1019-24.
41. Arrigo AP, Michel MR. Decreased heat-and tumor necrosis factor-mediated hsp28 phosphorylation in thermotolerant HeLa cells. FEBS Lett1991 Apr 22;282(1):152-6.
42. Gronborg M, Kristiansen TZ, Stensballe A, Andersen JS, Ohara O, Mann M, Jensen ON, Pandey A. A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies:identification of a novel protein, Frigg, as a protein kinase A substrate. Mol Cell Proteomics2002 Jul;1(7):517-27.
43. Steinberg TH, Agnew BJ, Gee KR, Leung WY, Goodman T, Schulenberg B, Hendrickson J, Beechem JM, Haugland RP, Patton WF. Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology. Proteomics2003 Jul;3(7):1128-44.
44. Wilkins CL, Weil DA, Yang CL, Ijames CF. High mass analysis by laser desorption Fourier transform mass spectrometry. Anal Chem1985 Feb;57(2):520-4.
45.许彬,魏开华,张学敏,杨松成.生物组织的基质辅助激光解吸电离质谱成像新进展.军事医学科学院院刊2006；30(3)：4.
46.刘念,魏开华,张学敏,杨松成.临床质谱学的最新进展：质谱成像方法及其应用.中国仪器仪表2007(10)：5.
[1]Brian G, Taylor H. Cataract blindness-challenges for the 21st century. Bull World Health Organ,2001,79(3):249-256.
[2]Quillen DA. Common causes of vision loss in elderly patients. Am Fam Physician, 1999,60(1):99-108.
[3]Mann M, Jensen ON. Proteomic analysis of post-translational modifications. Nat Biotechnol,2003,21(3):255-261
[4]Lapko VN, Cerny RL, Smith DL, et al. Modifications of human beta A1/beta A3-crystallins include S-methylation, glutathiolation, and truncation. Protein Sci 2005,14:45-54.
[5]Hains PG, Truscott RJW. Post-translational modifications in the nuclear region of young, aged, and cataract human lenses. J Proteome Res,2007,6:3935-3943.
[6]Wilmarth PA, Tanner S, Dasari S, et al. Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens:Does deamidation contribute to crystallin insolubility? J Proteome Res,2006,5:2554-2566.
[7]Takemoto L. Increased deamidation of asparagine-101 from alpha-A crystallin in the high molecular weight aggregate of the normal human lens. Exp. Eye Res,1999, 68:641±5.
[8]Takemoto L, Boyle D. Deamidation of alpha-A crystallin from nuclei of cataractous and normalhuman lenses. Mol. Vis,1999,5:2-8.
[9]Takumi T, Luke G, et al. Deamidation alters interactions of beta-crystallins in hetero-oligomers. Mol. Vis,2009,15(24):241-249
[10]Takata T, Oxford JT, et al. Deamidation destabilizes and triggers aggregation of a lens protein, beta A3-crystallin. Protein Sci,2008,17(9):1565-1575.
[11]Harms MJ, Wilmarth PA, Kapfer DM et al. Laser light-scattering evidence for an altered association of beta B1-crystallin deamidated in the connecting peptide. Protein Sci,2004,13(3):678-686.
[12]Michiel M, Duprat E, Feriel SP, et al. Aggregation of deamidated human beta B2-crystallin and incomplete rescue by alpha-crystallin chaperone. Exp. Eye Res, 2010,90(6):688-698.
[13]Lampi KJ, Kim YH, et al. Decreased heat stability and increased chaperone requirement of modified human gamma B1-crystallins..Mol. Vis,2002,8(37): 359-366.
[14]Flaugh SL, Mills IA, King J. et al. Glutamine deamidation destabilizes human gamma D-crystallin and lowers the kinetic barrier to unfolding. J Biol Chem,2006, 281(41):30782-30793.
[15]Chaves JM, Srivastava K, Gupta R,et al. Structural and functional roles of deamidation and/or truncation of N-or C-termini in human alpha A-crystallin. Biolchemistry,2008,47:10069-10083.
[16]Crabbe MJC, Cooper LR, Corne DW. Use of essential and molecular dynamics to study gamma B-crystallin unfolding after non-enzymic post-translational modifications. Comput Biol Chem,2003,27:507-510.
[17]Obrosova IG, Chung SS, Kador PF, et al. Diabetic cataracts:mechanisms and management.Diabetes-Metab Res,2010,26(3):172-180.
[18]Zarina S, Zhao HR, Abraham EC, et al. Advanced glycation end products in human senile and diabetic cataractous lenses.Mol Cell Biochem,2000,210(1-2): 29-34.
[19]Bhattacharyya J, ShipoVa EV, Santhoshkumar P,et al. Effect of a single AGE modification on the structure and chaperone activity of human alpha B-crystallin. Biochemistry,2007,46:14682-14692.
[20]Abraham EC, Jin HQ, Aziz A, et al. Role of the specifically targeted lysine residues in the glycation dependent loss of chaperone activity of alpha A-and alpha B-crystallins. Mol Cell Biochem,2008,310(1-2):235-239.
[21]Kumar PA, Kumar MS, Reddy GB. Effect of glycation on alpha-crystallin structure and chaperone-like function. Biochem.J,2007,408:251-258.
[22]Puttaiah S, Biswas A, Staniszewska M,et al. Methylglyoxal inhibits glycation-mediated loss in chaperone function and synthesis of pentosidine in alpha-crystallin. Exp Eye Res,2007,84:914-921.
[23]Truscott RJW, Augusteyn RC. The state of sulphydryl groups in normal and cataractous human lenses. Exp. Eye Res,1977c,Aug;25(2):139-48.
[24]Garner MH, Spector A. Selective oxidation of cysteine and methionine in normal and senile cataractous lenses. Proc. Nat1 Acad.Sci. USA 1980,77,1274-1277.139-148.
[25]Truscott RJW. Age-related nuclear cataract-oxidation is the key. Exp. Eye Res,2005,80(5):709-725.
[26]Sharma K.K, Santhoshkumar P. Lens aging:effects of crystallins. Biochim Biophys Acta,2009,1790:1095-1108.
[27]R ajan S, Horn C, Abraham EC. Effect of oxidation of alphaA-and alphaB-crystallins on their structure, oligomerization and chaperone function. Cell Biochem,2006,288:125-134
[28]Kantrow SP, Lee W, Sur A, et al. MSRA repairs lens cytochrome C and alpha-crystallin, Exp. Eye Res. (Supplement),2008,87:125.
[29]Molnar G.A, Nemes V, Biro Z,et al. Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase. Free Radical Res, 2005,39:1359-1366.
[30]Kanwar R, Balasubramanian D. Structure and stability of the dityrosine-linked dimer of gamma B-crystallin. Exp. Eye Res,1999,68:773-784.
[31]Kamei A, Hamaguchi T, Matsuura N, et al. Does post-translational modification influence chaperone-like activity ofalpha-crystallin? I. Study on phosphorylation. Biol. Pharm. Bull,2001,24(1):96-99.
[32]Fujii N, Ishibashi Y, Satoh K, et al. Simultaneous racemization and isomerization at specific aspartic acid residues in alpha-B-crystallin from the aged human lens. Biochim.Biophys.Acta,1994,1204:157-163.
[33]Lapko V.N, Smith D.L, Smith J.B. Methylation and carbamylation of human gamma-crystallins. Protein Sci,2003,12:1762-1774.
[34]Lund AL, Smith JB, Smith DL. Modifications of the water-insoluble human lens alpha-crystallins. Exp. Eye Res,1996,63(6):661-672.
[35]Ma Z, Hanso S.R, Lampi K, et al. Age-related changes in human lens crystallins identified by HPLC and mass spectrometry.Exp. Eye Res,1998,67:21±30.
[36]Srivastava O.P, Srivastava K, Silney C.Identification of a g-crystallin fragment in human lenses. Exp. Eye Res,1993,56:367±9.
[37]Srivastava K, Gupt a R,Chaves JM, et al. Truncated Human beta B1-Crystallin Shows Altered Structural Properties and Interaction with Human beta A3-Crystallin. Biochem,2009,48(30):7179-7189.
[38]Kamei A, Takeuchi N, Nagai M, et al. Post-translational modification of beta H-crystallin of bovine lens with aging. Biol Pharm Bull,2003,26:1715-1720.
[39]Lin PPP, Barry RC, Smith DL, et al. In vivo acetylation identified at lysine 70 of human lens alpha A-crystallin. Protein Sci,1998,7:1451-1457.
[40]Qin W, Smith JB, Smith DL. Reaction of aspirin with cysteinyl residues of lens y-crystallins:A mechanism for the proposed anti-cataract effect of aspirin. Biochim Biophys Acfa,1993,1181:103-110.
[41]Hong Yan, Yong Guo, Jie Zhang, et al. Effect of carnosine, aminoguanidine, and aspirin drops on the prevention of cataracts in diabetic rats. Mol.Vis,2008,14: 2282-2291
[42]Rao GN, Cotlier E. Aspirin prevents the nonenzymic glycosylation and carbamylation of the human eye lens crystallins in vitro. Biochem. Biophys. Res, 1988,151:991-996.
[43]Singha S,Bhattacharya J, Datta H, et al. Anti-glycation activity of gold nanoparticles. Nanomed Nanotech Biol Med,2009,5(1):21-29.
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35. Kamei A, Takamura S, Nagai M, Takeuchi N. Phosphoproteome analysis of hereditary cataractous rat lens alpha-crystallin. Biol Pharmacol Bull2004;27(12):1923-31.
36. Ueda Y, Duncan MK, David LL. Lens proteomics:The accumulation of crystallin modifications in the mouse lens with age. Invest Ophthalmol Vis Sci2002;43(1):205-15.
37. Ma Z, Hanson SR, Lampi KJ, David LL, Smith DL, Smith JB. Age-related changes in human lens crystallins identified by HPLC and mass spectrometry. Exp Eye Res1998 Jul;67(1):21-30.
38. Lampi KJ, Ma Z, Shih M, Shearer TR, Smith JB, Smith DL, David LL. Sequence analysis of betaA3, betaB3, and betaA4 crystallins completes the identification of the major proteins in young human lens. J Biol Chem 1997 Jan 24;272(4):2268-75.
39. Billingsley G, Santhiya ST, Paterson AD, Ogata K, Wodak S, Hosseini SM, Manisastry SM, Vijayalakshmi P, Gopinath PM, Graw J, Heon E. CRYBA4, a novel human cataract gene, is also involved in microphthalmia. Am J Hum Genet2006 Oct;79(4):702-9.
40. Zhou G, Zhou N, Hu S, Zhao L, Zhang C, Qi Y. A missense mutation in CRYBA4 associated with congenital cataract and microcornea. Mol Vis2010;16:1019-24.
41. Arrigo AP, Michel MR. Decreased heat-and tumor necrosis factor-mediated hsp28 phosphorylation in thermotolerant HeLa cells. FEBS Lett1991 Apr 22;282(1):152-6.
42. Gronborg M, Kristiansen TZ, Stensballe A, Andersen JS, Ohara O, Mann M, Jensen ON, Pandey A. A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies:identification of a novel protein, Frigg, as a protein kinase A substrate. Mol Cell Proteomics2002 Jul;1(7):517-27.
43. Steinberg TH, Agnew BJ, Gee KR, Leung WY, Goodman T, Schulenberg B, Hendrickson J, Beechem JM, Haugland RP, Patton WF. Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology. Proteomics2003 Jul;3(7):1128-44.
44. Wilkins CL, Weil DA, Yang CL, Ijames CF. High mass analysis by laser desorption Fourier transform mass spectrometry. Anal Chem1985 Feb;57(2):520-4.
45.许彬,魏开华,张学敏,杨松成.生物组织的基质辅助激光解吸电离质谱成像新进展.军事医学科学院院刊2006；30(3)：4.
46.刘念,魏开华,张学敏,杨松成.临床质谱学的最新进展：质谱成像方法及其应用.中国仪器仪表2007(10)：5.
[1]Brian G, Taylor H. Cataract blindness-challenges for the 21st century. Bull World Health Organ,2001,79(3):249-256.
[2]Quillen DA. Common causes of vision loss in elderly patients. Am Fam Physician, 1999,60(1):99-108.
[3]Mann M, Jensen ON. Proteomic analysis of post-translational modifications. Nat Biotechnol,2003,21(3):255-261
[4]Lapko VN, Cerny RL, Smith DL, et al. Modifications of human beta A1/beta A3-crystallins include S-methylation, glutathiolation, and truncation. Protein Sci 2005,14:45-54.
[5]Hains PG, Truscott RJW. Post-translational modifications in the nuclear region of young, aged, and cataract human lenses. J Proteome Res,2007,6:3935-3943.
[6]Wilmarth PA, Tanner S, Dasari S, et al. Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens:Does deamidation contribute to crystallin insolubility? J Proteome Res,2006,5:2554-2566.
[7]Takemoto L. Increased deamidation of asparagine-101 from alpha-A crystallin in the high molecular weight aggregate of the normal human lens. Exp. Eye Res,1999, 68:641±5.
[8]Takemoto L, Boyle D. Deamidation of alpha-A crystallin from nuclei of cataractous and normalhuman lenses. Mol. Vis,1999,5:2-8.
[9]Takumi T, Luke G, et al. Deamidation alters interactions of beta-crystallins in hetero-oligomers. Mol. Vis,2009,15(24):241-249
[10]Takata T, Oxford JT, et al. Deamidation destabilizes and triggers aggregation of a lens protein, beta A3-crystallin. Protein Sci,2008,17(9):1565-1575.
[11]Harms MJ, Wilmarth PA, Kapfer DM et al. Laser light-scattering evidence for an altered association of beta B1-crystallin deamidated in the connecting peptide. Protein Sci,2004,13(3):678-686.
[12]Michiel M, Duprat E, Feriel SP, et al. Aggregation of deamidated human beta B2-crystallin and incomplete rescue by alpha-crystallin chaperone. Exp. Eye Res, 2010,90(6):688-698.
[13]Lampi KJ, Kim YH, et al. Decreased heat stability and increased chaperone requirement of modified human gamma B1-crystallins..Mol. Vis,2002,8(37): 359-366.
[14]Flaugh SL, Mills IA, King J. et al. Glutamine deamidation destabilizes human gamma D-crystallin and lowers the kinetic barrier to unfolding. J Biol Chem,2006, 281(41):30782-30793.
[15]Chaves JM, Srivastava K, Gupta R,et al. Structural and functional roles of deamidation and/or truncation of N-or C-termini in human alpha A-crystallin. Biolchemistry,2008,47:10069-10083.
[16]Crabbe MJC, Cooper LR, Corne DW. Use of essential and molecular dynamics to study gamma B-crystallin unfolding after non-enzymic post-translational modifications. Comput Biol Chem,2003,27:507-510.
[17]Obrosova IG, Chung SS, Kador PF, et al. Diabetic cataracts:mechanisms and management.Diabetes-Metab Res,2010,26(3):172-180.
[18]Zarina S, Zhao HR, Abraham EC, et al. Advanced glycation end products in human senile and diabetic cataractous lenses.Mol Cell Biochem,2000,210(1-2): 29-34.
[19]Bhattacharyya J, ShipoVa EV, Santhoshkumar P,et al. Effect of a single AGE modification on the structure and chaperone activity of human alpha B-crystallin. Biochemistry,2007,46:14682-14692.
[20]Abraham EC, Jin HQ, Aziz A, et al. Role of the specifically targeted lysine residues in the glycation dependent loss of chaperone activity of alpha A-and alpha B-crystallins. Mol Cell Biochem,2008,310(1-2):235-239.
[21]Kumar PA, Kumar MS, Reddy GB. Effect of glycation on alpha-crystallin structure and chaperone-like function. Biochem.J,2007,408:251-258.
[22]Puttaiah S, Biswas A, Staniszewska M,et al. Methylglyoxal inhibits glycation-mediated loss in chaperone function and synthesis of pentosidine in alpha-crystallin. Exp Eye Res,2007,84:914-921.
[23]Truscott RJW, Augusteyn RC. The state of sulphydryl groups in normal and cataractous human lenses. Exp. Eye Res,1977c,Aug;25(2):139-48.
[24]Garner MH, Spector A. Selective oxidation of cysteine and methionine in normal and senile cataractous lenses. Proc. Nat1 Acad.Sci. USA 1980,77,1274-1277.139-148.
[25]Truscott RJW. Age-related nuclear cataract-oxidation is the key. Exp. Eye Res,2005,80(5):709-725.
[26]Sharma K.K, Santhoshkumar P. Lens aging:effects of crystallins. Biochim Biophys Acta,2009,1790:1095-1108.
[27]R ajan S, Horn C, Abraham EC. Effect of oxidation of alphaA-and alphaB-crystallins on their structure, oligomerization and chaperone function. Cell Biochem,2006,288:125-134
[28]Kantrow SP, Lee W, Sur A, et al. MSRA repairs lens cytochrome C and alpha-crystallin, Exp. Eye Res. (Supplement),2008,87:125.
[29]Molnar G.A, Nemes V, Biro Z,et al. Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase. Free Radical Res, 2005,39:1359-1366.
[30]Kanwar R, Balasubramanian D. Structure and stability of the dityrosine-linked dimer of gamma B-crystallin. Exp. Eye Res,1999,68:773-784.
[31]Kamei A, Hamaguchi T, Matsuura N, et al. Does post-translational modification influence chaperone-like activity ofalpha-crystallin? I. Study on phosphorylation. Biol. Pharm. Bull,2001,24(1):96-99.
[32]Fujii N, Ishibashi Y, Satoh K, et al. Simultaneous racemization and isomerization at specific aspartic acid residues in alpha-B-crystallin from the aged human lens. Biochim.Biophys.Acta,1994,1204:157-163.
[33]Lapko V.N, Smith D.L, Smith J.B. Methylation and carbamylation of human gamma-crystallins. Protein Sci,2003,12:1762-1774.
[34]Lund AL, Smith JB, Smith DL. Modifications of the water-insoluble human lens alpha-crystallins. Exp. Eye Res,1996,63(6):661-672.
[35]Ma Z, Hanso S.R, Lampi K, et al. Age-related changes in human lens crystallins identified by HPLC and mass spectrometry.Exp. Eye Res,1998,67:21±30.
[36]Srivastava O.P, Srivastava K, Silney C.Identification of a g-crystallin fragment in human lenses. Exp. Eye Res,1993,56:367±9.
[37]Srivastava K, Gupt a R,Chaves JM, et al. Truncated Human beta B1-Crystallin Shows Altered Structural Properties and Interaction with Human beta A3-Crystallin. Biochem,2009,48(30):7179-7189.
[38]Kamei A, Takeuchi N, Nagai M, et al. Post-translational modification of beta H-crystallin of bovine lens with aging. Biol Pharm Bull,2003,26:1715-1720.
[39]Lin PPP, Barry RC, Smith DL, et al. In vivo acetylation identified at lysine 70 of human lens alpha A-crystallin. Protein Sci,1998,7:1451-1457.
[40]Qin W, Smith JB, Smith DL. Reaction of aspirin with cysteinyl residues of lens y-crystallins:A mechanism for the proposed anti-cataract effect of aspirin. Biochim Biophys Acfa,1993,1181:103-110.
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