由老化或其他疾病所造成的各種眼睛疾病中，白內障是相當普遍的一種。為了對白內障發生的原因及發病過程有更進一步的了解，利用二維電泳搭配高解析度質譜技術與高效能液相層析系統搭配高解析度質譜技術兩種方法來研究由人類糖尿病型白內障水晶體的水溶性蛋白質及尿素可溶性蛋白質。二維之電泳實驗結果發現，在水溶性蛋白部分，分子量約50-60kDa的地方有蛋白質聚集體，共有Alpha-crystallin A、Beta-crystallin B1、B2、A3、A4、S還有Gamma-crystallin A與D共八種水晶體蛋白的胜肽片段，由此推測這八種水晶體蛋白在老化的過程中會因為一些後轉譯修飾造成共價聚集的現象。由糖化之研究實驗結果發現，在水溶性蛋白部分以及尿素可溶性蛋白部分中都含有兩種最終糖化蛋白(Advanced Glycation End Products，AGEs)產生，分別是Nε-carboxymethyllysine (CML)與Oxalate monoalkylamide，並由一次及二次質譜證明糖化修飾位置分別是Alpha-crystallin A的M1、Alpha-crystallin B的M1、Beta-crystallin B1的K118、Beta-crystallin A3的K131以及Beta-crystallin S的K7及K159。此實驗結果證實了水晶體中最終糖化蛋白修飾的存在，導致其結構之變化而變性沉澱進而促進白內障之生成。

Cataract is a common disease which is caused mainly due to aging, diabetes, or foreign stresses. In order to explore further regarding to how the diabetes effect the aging diabetic lens crystallins, proteomics techniques were employed. The water-soluble protein fraction and the urea-soluble protein fraction extracted from human diabetic cataract lens were performed the two-dimensional gel electrophoresis followed by high resolution mass spectrometry (LTQ Orbitrap XL) analysis. It was found that there was a protein point cluster with the molecular weight around 60-70kDa showing multiple aggregates, which consisted of eight crystallins, including Alpha-crystallin A, Beta-crystallin B1,B2,A3,A4,S, and Gamma-crystallin A,D. This result suggested that aging diabetic lens underwent post translational modification as to change its structure, which in turn leading to the formation of cataract. By using the ultraperformance liquid chromatography followed by high resolution tandem mass spectrometry, two kind of advanced glycation end products(AGEs), Nε-carboxymethyllysine (CML) and Oxalate monoalkylamide were observed in both water-soluble protein fraction and urea-soluble protein fraction. The modification sites were at methionine 1 in both Alpha-crystallin A and Alpha-crystallin B, lycine118 in Beta-crystallin B1, lycine 131 in Beta-crystallin A3, and lycine7 and 159 in Beta-crystallin S. This result confirmed the existence of advanced glycation end products in diabetic human lens, and also suggested that those modifications might affect the function and structure of crystallins in human lens.

[1] 張孟媛, 失去光明的水晶：淺談白內障, 科學EasyLeran網路版, 醫學,
2012.
[2] J. Horwitz, Alpha-crystallin, Experimental Eye Research, 76, 145-153,
2003.
[3] J. Horwitz, M.P. Bova, L.L. Ding, D.A. Haley, P.L Stewart , Lens
alpha-crystallin: function and structure, Eye (Lond). Pt 3b, 403-408, 1999.
[4] A. Spector, D. Roy, Disulfide-linked high molecular weight protein
associated with human cataract, Biochem, 75, 3244-3248, 1978.
[5] A. Abbasi, L.S. David, B.S. Jean, Characterization of low molecular mass
γ-crystallin fragments from juman lenses, Exp. Eye Res., 67, 485-488,
1998.
[6] E.S. Lander, et al, Initial sequencing and analysis of the human genome,
Nature, 409, 860-921, 2001.
[7] V.C. Wasinger, S.J. Cordwell, A. Cerpa-Poljak, J.X. Yan, A.A. Gooley,
M.R. Wilkins, M.W. Duncan, R. Harris, K.L. Williams, I.
Humphery-Smith, Progress with gene-product mapping of the Mollicutes:
Mycoplasma genitalium, Electrophoresis, 16, 1090-1094, 1995.
[8a] B.S. Hartley, Strategy and Tactics in Protein Chemistry - First Bdh
Lecture, Biochemical Journal, 119, 5, 805-822, 1970.
[8b] Liebler, D.C., Introduction to Proteomics : Tools for the New Biology,
2001.
[9] 陳玉如, 質譜技術與蛋白質體學, Mass Spectrometry and Proteomics,
第三章.
[10] S.A. Hofstadler, K. A. Sannes-Lowery, Applications of ESI-MS in drug
discovery: interrogation of noncovalent complexes, Nature Reviews
Drug Discovery, 5, 585-595, 2006.
[11] M. Karas, D. Bachmann, U. Bahr, F. Hillenkamp, Matrix-assisted
ultraviolet laser desorption of non-volatile compounds, International
Journal of Mass Spectrometry and Zon Processes, 78, 53-68, 1987.
[12] G.L. Glish, R. W.Vachet, The basics of mass spectrometry in the
twentyfirst century, Nature Reviews Drug Discovery, 2, 140-150, 2003.
[13] Thermo Electron (Bremen) GmbH, Bremen, Germany, Dynamic Range
of Mass Accuracy in LTQ Orbitrap Hybrid Mass Spectrometer, Journal
of the American Society for Mass Spectrometry, 17, 7, 977–982, 2006.
[14] J. Harding, Cataract: biochemistry, epidemiology and pharmacology.
Chapman & Hall, London, 1991.
[15] 林佳葳, 吳思緯, 于心宜, 邱繼輝,質譜分析技術之應用於醣質體
學,Chemistry (The Chinese Chemical Society, Taipei), June 2007,
125-136
[16a] R. Singh, A. Barden, T. Mori, L. Beilin, Advanced glycation
end-products: a review, Diabetologia 44, 129-146, 2001.
[16b] N. Ahmed, Advanced glycation endproducts—role in pathology of
diabetic complications, Diabetes Res. Clin. Pract, 67, 3-21, 2005.
[17] H.T. Beswick, J.J. Harding, Conformational changes induced in lens
alpha- and gamma-crystallins by modification with glucose 6-phosphate.
Implications for cataract, Biochem. J., 246, 761-769, 1987.
[18] R. McNulty, H. Wang, R.T. Mathias, B.J. Ortwerth, R.J.W. Truscott, S.
Bassnett, Regulation of tissue oxygen levels in the mammalian lens, J
Physiol, 559, 883-898, 2004.
[19] Y.B. Shui, D.C. Beebe, Age-dependent control of lens growth by
hypoxia, Invest Ophthalmol Vis Sci, 49, 1023–1029, 2008.
[20] M. Tomana, J.T. Prchal, L.C. Garner, H.W.Skalka, S.A. Barker, Gas
chromatographic analysis of lens monosaccharides, J Lab Clin Med, 103,
137-142, 1984.
[21] V.M. Monnier, A. Cerami, Nonenzymatic browning in vivo: possible
process for aging of long-lived proteins, Science, 211, 491-493, 1981.
[22] R.L. Garlick, J.S. Mazer, L.T. Chylack, Jr, W.H. Tung, H.F. Bunn,
Nonenzymatic glycation of human lens crystallin effect of aging and
diabetesmellitus, J Clin Invest, 74, 1742-1749, 1984.
[23] M. Oimomi, Y. Maeda, F. Hata F, Y. Kitamura, S. Matsumoto, S.
Baba, T. Iga, M. Yamamoto, Glycation of cataractous lens in
non-diabetic senile subjects and in diabetic patients, Exp Eye Res., 46,
415-420, 1988.
[24] M. Yano, S. Matsuda, Y. Bando, K. Shima, Lens protein glycation and
the subsequent degree of opacity in streptozotocin-diabetic rats, Diabetes
Res Clin Pract, 7, 259-262, 1989.
[25] M. Larsen, B. Kjer, I. Bendtson, P. Dalgaard, H. Lund-Andersen, Lens
fluorescence in relation to metabolic control of insulin-dependent
diabetes mellitus, Arch Ophthalmol, 107, 59-62, 1989.
[26] J.N. Liang, Fluorescence study of the effects of aging and diabetes
mellitus on human lens alpha-crystallin, Curr Eye Res, 6, 351-355, 1987.
[27] M. Lutze, G.H Bresnick, Lenses of diabetic patients ‘‘yellow’’ at an
accelerated rate similar to older normal, Invest Ophthalmol Vis Sci, 32,
194–199, 1997.
[28] M.U. Ahmed, S.R. Thorpe, J.W Baynes, Identification of N
epsilon-carboxymethyllysine as a degradation product of fructoselysine
in glycated protein, J Biol Chem, 261, 4889–4894, 1986.
[29] S. Franke, J. Dawczynski, J. Strobel, T. Niwa, P. Stahl, G. Stein,
Increased levels of advanced glycation end products in human
cataractous lenses, J Cataract Refract Surg, 29, 998-1004, 2003.
[30] T.J. Lyons, G. Silvestri, J.A. Dunn, D.G. Dyer, J.W. Baynes, Role of
glycation in modification of lens crystallins in diabetic and nondiabetic
senile cataracts, Diabetes 40, 1010-1015, 1991.
[31] F. Tessier, M. Obrenovich, V.M Monnier, Structure and mechanism of
formation of human lens fluorophore LM-1. Relationship to vesperlysine
A and the advanced Maillard reaction in aging, diabetes, and
cataractogenesis, J Biol Chem, 274, 20796-20804, 1999.
[32] D.R. Sell, V.M. Monnier, Structure elucidation of a senescence
cross-link from human extracellular matrix. Implication of pentoses in
the aging process, J Biol Chem, 264, 21597-21602, 1989.
[33] B.J. Ortwerth, P.R Olesen, Ascorbic acid-induced crosslinking of lens
proteins: evidence supporting a Maillard reaction, Biochim Biophys Acta
956, 10-22, 1988.
[34] R.H. Nagaraj, D.R. Sell, M. Prabhakaram, B.J. Ortwerth, V.M. Monnier,
High correlation between pentosidine protein crosslinks and
pigmentation implicates ascorbate oxidation in human lens senescence
and cataractogenesis, Proc Natl Acad Sci USA, 88, 10257-10261, 1991.
[35] S. Reddy, J. Bichler, K.J. Wells-Knecht, S.R. Thorpe, J.W. Baynes, N
epsilon- (carboxymethyl)lysine is a dominant advanced glycation end
product (AGE) antigen in tissue proteins, Biochemistry, 34,
10872-10878, 1995.
[36] R.H. Nagaraj, V.M. Monnier, Isolation and characterization of a blue
fluorophore from human eye lens crystallins: in vitro formation from
Maillard reaction with ascorbate and ribose, Biochim Biophys Acta,
1116, 34-42, 1992.
[37] Ramanakoppa H. Nagaraja;b, Farrukh A. Shamsia, Birgit Huberc,
Monika Pischetsriederc, Immunochemical detection of oxalate
monoalkylamide, an ascorbate-derived Maillard reaction product in the
human lens, FEBS LETTER, 453(3), 1999, pp. 327-330
[38] A. Spector, The search for a solution to senile cataracts, Proctor lecture,
Invest Ophthalmol Vis Sci, 25, 130-146, 1984.
[39] R.H. Nagaraj, D.R. Sell, M. Prabhakaram, B.J. Ortwerth, V.M. Monnier,
High correlation between pentosidine protein crosslinks and
pigmentation implicates ascorbate oxidation in human lens senescence
and cataractogenesis, Proc Natl Acad Sci USA, 88, 10257-10261, 1991.
[40] M. McDermott, R. Chiesa, J.E Roberts, J. Dillon, Photooxidation of
specific residues in alpha-crystallin polypeptides, Biochemistry, 30,
8653-8660, 1991.
[41] M.J. MacCoss, W.H. McDonald, A. Saraf, R. Sadygov, J.M Clark, J.J.
Tasto, K.L. Gould, D. Wolters, M. Washburn, A. Weiss, J.I. Clark, J.R.
3rd Yates, Shotgun identification of protein modifications from protein
complexes and lens tissue, Proc Natl Acad Sci USA, 99, 7900-7905,
2002.
[42] P.A. Wilmarth, S. Tanner, S. Dasari, S.R. Nagalla, M.A. Riviere, V.
Bafna, P.A. Pevzner, L.L. David, 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, 5, 2554-2566, 2006.
[43] R. Gupta, O.P. Srivastava, Effect of deamidation of asparagine 146 on
functional and structural properties of human lens alphaB-crystallin,
Invest Ophthalmol Vis Sci, 45,206-214, 2004.
[44] O.P. Srivastava, K. Srivastava, Existence of deamidated alphaB-crystallin
fragments in normal and cataractous human lenses, Mol Vis , 9, 110-118,
2003.
[45] P.P. Lin, R.C. Barry, D.L. Smith, J.B. Smith, In vivo acetylation
identified at lysine 70 of human lens alphaA-crystallin, Protein Sci, 7,
1451-1457, 1998.
[46] Y. Ueda, M.K Duncan, L.L. David, Lens proteomics: the accumulation of
crystallin modifications in the mouse lens with age, Invest Ophthalmol
Vis Sci, 43, 205-215, 2002.
[47] P.A. Kumar, M.S. Kumar, G. Bhanuprakash Reddy, Effect of glycation
on α-crystallin structure and chaperone-like function, Biochem J,
408(Pt2), 251-258, 2007.
[48] J.F. Hess, J.T. Casselman, A.P. Kong, P.G. Fitzgerald, Primary sequence,
secondary structure, gene structure, and assembly properties suggests
that the lens-specific cytoskeletal protein filensin represents a novel class
of intermediate filament protein, Exp. Eye Res, 66, 625-644, 1998.
[49] P.G. FitzGerald, W. Gottlieb W, The 115-kd fiber cell specific protein is
a component of the lens cytoskeleton, Curr Eye Res, 8, 801–811, 1989.
[50] O.P. Srivastava, M.C Kirk, K. Srivastava, Characterization of Covalent
Multimers of Crystallins in Aging Human Lenses, J Biol Chem, 279,
10901-10909, 2004.
[51] V. Harrington, S. McCall, S. Huynh, K. Srivastava, O.P. Srivastava,
Crystallins in water soluble-high molecular weight protein fractions and
water insoluble protein fractions in aging and cataractous human lenses,
Mol Vis, 10, 476-489, 2004.