糖尿病性白内障晶状体蛋白二维电泳和质谱鉴定

目的应用二维电泳和质谱鉴定技术对糖尿病性白内障进行蛋白质组学研究,探讨蛋白质组学在糖尿病性白内障发病机制中的价值。方法取8例(8眼)1型糖尿病并发白内障晶状体组织,提取晶状体组织中蛋白质进行二维电泳,凝胶染色后,通过Gel Doc2000凝胶成像仪采集图像,利用PDQuest8.0凝胶分析软件对图像进行分析。使用基质辅助激光分析离子飞行时间质谱仪(MALAI-TOF-MS)对蛋白斑点进行鉴定。结果二维电泳图谱能检测到35～40个左右的蛋白斑点,大部分高丰度蛋白斑点相对分子质量处于(20～31)×103范围内,小部分低丰度蛋白斑点相对分子质量处于(35～45)×103范围内,等电点在5～9之间。利用MASCOT软件查询NCBInr数据库对2个蛋白斑点的肽质量指纹图谱进行了鉴定,分别为α-B晶状体蛋白和β-B1晶状体蛋白。结论二维电泳和质谱鉴定能够较好地分离和鉴定糖尿病性白内障晶状体蛋白质,为研究糖尿病性白内障形成机制提供了新的方法和途径。     

先天性白内障患者和正常人晶状体蛋白质组的差异分析

目的 鉴定并分析先天性白内障患者和正常人晶状体之间蛋白质表达的差异.方法 取10例(20眼)先天性白内障晶状体及8例(8眼)正常透明晶状体,提取晶状体核中的水溶性蛋白行二维电泳及考马斯亮蓝凝胶染色后,用Image Master 2D Platinum 5.0软件对获得的蛋白图谱进行分析,再利用MALDI-TOF-MS分析及数据库搜索对差异表达的蛋白质点进行鉴定.ELISA法检测差异蛋白的含量.结果 二维电泳结果显示,先天性白内障和正常晶状体蛋白均分布在相对分子质量为14 400 ～97 400、pI5～9区域内;高丰度蛋白质斑点分布在相对分子质量20000 ～ 31000、pI 6 ～8区域内.正常透明晶状体核水溶性蛋白二维电泳图识别出34个蛋白质斑点,先天性白内障蛋白二维电泳图识别出31个点有4个.对选取的蛋白质差异斑点进行MALDI-TOF-MS分析,经数据库检索后鉴定出4种蛋白质,分别为βA3晶状体蛋白、βB1晶状体蛋白、截断的βB1晶状体蛋白和αB晶状体蛋白.ELISA结果显示,正常晶状体βA3、αB和βB1晶状体蛋白质量浓度分别为(2.20±0.15)g·L-1、(0.85±0.08)g·L-1、(0.72±0.05)g·L-1,而先天性白内障βA3、αB和βB1晶状体蛋白质量浓度为(1.60±0.09)g·L-1、(1.02±0.09)g·L-1、(0.59±0.07)g·L-1.先天性白内障αB晶状体蛋白较正常对照含量上调,βA3和βB1晶状体蛋白含量下调.结论 αB晶状体蛋白上调、βA3和βB1晶状体蛋白含量下调可能与先天性白内障的发病相关.     

白内障晶状体核蛋白质组学的初步研究

目的研究年龄相关性白内障晶状体核不同组分晶状体蛋白的组成。方法取12例年龄相关性白内障晶状体软核,提取晶状体核中的水溶性蛋白及尿溶性蛋白进行二维电泳及UPD-蛋白质染料染色试剂盒对凝胶染色后,用Image Master2D Platinum6.0软件对获得的蛋白图谱进行分析,再利用MALDI-TOF MS分析及数据库搜索对差异表达的蛋白质点进行鉴定。结果二维电泳结果显示,年龄相关性白内障晶状体核中水溶性蛋白和尿溶性蛋白大部分为相对分子质量14400~97400,pI5~9。高丰度蛋白质斑点相对分子质量分布在20000~31000,pI6~8区域内。年龄相关性白内障晶状体核水溶性蛋白二维电泳图识别出31个蛋白质斑点,尿溶性蛋白二维电泳图识别出26个蛋白质斑点,相差2倍以上表达的差异点有14个。对选取的蛋白质斑点进行MALDI-TOF MS分析,经数据库检索后鉴定出6种蛋白质,其中3种蛋白质表达下调（γS-晶状体蛋白、βA3-晶状体蛋白、βA4-晶状体蛋白）,1种蛋白质表达上调（βB1-晶状体蛋白）,2种蛋白质表达缺失（截取的人βB1-晶状体蛋白A链、截取的人γD-晶状体蛋白X链）。结论年龄相关性白内障晶状体核不同组分晶状体蛋白中主要由β-晶状体蛋白组成,与尿溶性晶状体蛋白相比,水溶性蛋白鉴定出γD-晶状体蛋白,而βA4-晶状体蛋白阙如。     

兔晶状体蛋白质组的双向电泳和质谱鉴定研究

目的探讨双向电泳和质谱鉴定在晶状体蛋白质组特性研究中的价值,为白内障的防治提供新的有效途径。方法采用固相pH梯度(IPG)等电聚焦双向凝胶电泳方法对兔龄为3个月的新西兰兔透明晶状体蛋白质进行分离,使用ImageMaster 2D Elite3．01图像分析软件分析电泳图像,使用基质辅助激光解吸附飞行时间质谱仪对主要的高丰度晶状体蛋白质进行鉴定。结果双向电泳图谱显示,在250μg兔晶状体中蛋白质分布在等电点(PI)为pH值5～9、相对分子质量为14000～94000的区域内;而高丰度晶状体蛋白质的相对分子质量为14000～40000。图像分析软件定量检出180个蛋白质斑点的近似PI、相对分子质量及蛋白质相对含量,质谱鉴定得到其中16个高丰度晶状体蛋白质的种类。结论双向电泳和质谱鉴定可有效分离和分析晶状体蛋白质组的特性,为分析白内障形成过程中蛋白质的表达改变提供了新的方法和途径,为白内障的防治带来了新的前景。     

先天性全白内障晶状体和透明晶状体蛋白双向电泳差异比较

目的 应用双向电泳（2-DE）技术分析先天性全白内障晶状体和正常透明晶状体蛋白质组成的差异.方法 采用固相pH梯度（IPG）等电聚焦2-DE技术分离全白内障组和正常对照组的晶状体蛋白后,比较两组蛋白质的组成差异.结果 人眼晶状体蛋白的主要分布在等电点（pI）5～9,相对分子质量14 000～90 000的范围内,高丰度的蛋白主要集中在pI 6～8,相对分子质量14 000～30 000之间.在pI 5.0～7.25,相对分子质量50 000～80 000的高分子量区间全白内障组具有较多的蛋白表达,在pI 6.7～7.7,相对分子质量19 000～29 000间的区域,全白内障组的部分正常蛋白点消失,在pI 7.1～7.6,相对分子质量15 000～17 000区间存在一些新的小分子量的蛋白点.结论 应用2-DE技术能够较好地分离人眼晶状体蛋白,全白内障组高分子量的蛋白质成分明显多于正常对照组,全白内障组的部分正常晶状体蛋白发生缺失,出现新的小分子量的蛋白成分.     

年龄相关性白内障和正常晶状体蛋白质组的差异分析

目的 运用双向电泳和基质辅助的激光解吸电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF-MS）的方法,鉴定年龄相关性白内障和正常晶状体之间差异表达的蛋白质.方法 用固相pH梯度双向凝胶电泳技术分离16例年龄相关性白内障和正常晶状体之间的总蛋白质,建立二维电泳图谱,再利用MALDI-TOF-MS分析及数据库搜索相结合对差异表达的蛋白质点进行鉴定.结果 双向电泳胶图显示年龄相关性白内障和正常晶状体共有84个差异点,其中52个上调表达,32个下调表达.有和无的差异表达点10个,相差3倍以上差异表达点10个.利用MALDI-TOF-MS对差异表达的15个蛋白质点进行肽指纹图谱分析,经数据库检索后鉴定出5个蛋白质.结论 年龄相关性白内障和正常晶状体之间蛋白表达存在差异,蛋白质组的分析结果为年龄相关性白内障的基础研究提供依据和新实验方法.     

运用双向荧光差异凝胶电泳分析人类年龄相关性核性白内障和正常晶状体核的蛋白质组学差异

目的 鉴定年龄相关性核性白内障(age-related nuclear cataract,ARNC)和正常晶状体核蛋白质组学差异.方法 用强解聚试剂分别溶解7只不同程度ARNC晶状体核和7只正常晶状体核,提取总蛋白,Bradford法测定蛋白浓度.通过双向荧光差异凝胶电泳(two-dimensional fluorescence difference in-gel electrophoresis,2-D DIGE)、Decyder 6.5软件分析电泳图谱,寻找差异蛋白质点.差异蛋白质点随后用激光辅助解吸/电离飞行时间(matrix-assisted laser desorption/ionization time-of-flight,MALDI-TOF)质谱仪鉴定.结果 2-D DIGE结果显示,和正常晶状体核样品相比,39个点的蛋白含量在ARNC样品中显著减少(P＜0.05).MALDI-TOF质谱成功鉴定其中36个蛋白质点,它们分属于9个不同的蛋白质,其中6个蛋白(αA-、βA3-、βA4-、βB1-、γD-晶状体蛋白和一个来自CRYGS家族的未知蛋白DKFZp434A0627)的含量随着ARNC程度的增加而逐渐减少；其他3种蛋白(αB-晶状体蛋白、3-磷酸甘油醛脱氢酶和视黄醛脱氢酶1)未呈现这种递减趋势.结论 3-磷酸甘油醛脱氢酶、视黄醛脱氢酶1以及某些晶状体蛋白的减少可能参与ARNC的形成.     

年龄相关性核性白内障晶状体核中不可逆聚集体的定性和定量分析

目的 鉴定分析在年龄相关性核性白内障(age-related nuclear cataract,ARNC)晶状体核中发现的不可逆聚集体的蛋白质组成.方法 用强解聚试剂和促溶剂溶解ARNC晶状体核(8例)和年龄匹配的正常晶状体核(8例),提取总蛋白,经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分离,在ARNC晶状体样品中发现的相对分子质量高的聚集体进行液相色谱串联质谱(LC-MS/MS)鉴定分析,确定聚集体的组成成分,并根据Mascot搜索数据估算各成分的相对含量.结果 SDS-PAGE结果显示,与正常晶状体核相比,ARNC样品在相对分子质量≤20 000处蛋白含量显著减少(均为P＜0.05),但在相对分子质量高(＞200 000)处蛋白质含量显著增加(均为P＜0.01).在ARNC晶状体内存在不可逆的相对分子质量高的聚集体.LC-MS/MS分析显示,这些聚集体主要来源于各种晶状体蛋白,同时还包括Filensin、Phakinin和碳酰还原酶1.在所有组分中αA-晶状体蛋白含量最高(19.1％).αA-、αB-、γD-晶状体蛋白和DKFZp434A0627比其他晶状体蛋白更容易聚集.结论 晶状体蛋白,尤其是αA-晶状体蛋白在白内障产生和发展过程中发生不可逆的聚集,细胞骨架蛋白Filensin和Phakinin以及碳酰还原酶1可能参与和加速这一过程.     

囊袋阻滞综合征患者囊袋内积聚液体及后发性白内障囊膜的蛋白质组成分析

目的　鉴定白内障术后囊袋阻滞综合征（capsular block syndrome,CBS）患者囊袋内积聚液体以及后发性白内障囊膜的蛋白质组成。方法　选取我院就诊的5例白内障超声乳化吸出联合人工晶状体植入术后CBS患者,继发青光眼且合并后发性白内障,吸出人工晶状体和后囊之间积聚的液体,撕除混浊的后发性白内障囊膜,用强裂解液提取蛋白质,进行液相色谱串联质谱鉴定分析,确定蛋白质组成,并根据Mascot搜索数据结果估算各成分的相对含量。所得蛋白质组成数据与同龄白内障患者晶状体皮质和晶状体上皮细胞的蛋白质主要组成进行比较及分析。结果　本研究中CBS患者人工晶状体和后囊之间积聚的液体和后发性白内障囊膜蛋白质组成中,含量较高（前10种）有细胞骨架类蛋白:晶纤蛋白、肌动蛋白、波形蛋白、锚蛋白2;晶状体蛋白:αA-晶状体蛋白、βB1-晶状体蛋白、αB-晶状体蛋白;葡萄糖代谢相关蛋白:三磷酸甘油醛脱氢酶、促进性葡萄糖转运蛋白;热休克蛋白A5。晶状体上皮细胞含量丰富的蛋白质（前15位）包含上述成分中的αA-晶状体蛋白、αB-晶状体蛋白、波形蛋白、肌动蛋白以及三磷酸甘油醛脱氢酶;而晶状体皮质检测到的含量较高的蛋白主要是各种晶状体蛋白,包含CBS患者囊袋内积液和后发性白内障囊膜组成中的βB1-晶状体蛋白、αA-晶状体蛋白、αB-晶状体蛋白。结论　CBS积聚的液体和后发性白内障囊膜主要蛋白质组成与晶状体上皮细胞更加相似,它们可能参与CBS囊袋内液体的积聚以及此种后发性白内障的形成。     

SIRT1基因在糖尿病性白内障患者晶状体上皮细胞表达的初步研究

目的:探讨SIRT1基因在糖尿病性白内障晶状体上皮细胞的表达.方法:选取2012-01/2014-10来我院治疗的糖尿病性白内障患者、年龄相关性白内障患者和外伤性白内障患者各20例,采用RT-PCR法检测各组患者晶状体上皮细胞中SIRT1基因含量,Western blot法检测晶状体上皮细胞中SIRT1蛋白含量,TUNEL法检测晶状体上皮细胞的凋亡率.结果:RT-PCR检测结果显示,外伤性白内障患者组SIRT1 mRNA相对含量最高为1.000±0.078,其次为年龄相关性白内障患者组为0.427±0.067,糖尿病性白内障组为0.389±0.112,与外伤性白内障组比较,差异有统计学意义(P＜0.05);Western blot检测显示,外伤性白内障患者晶状体上皮细胞中SIRT1蛋白的表达量最高,其次是年龄相关性白内障组,糖尿病性白内障组SIRT1蛋白的表达量最低;TUNEL法检测结果显示,外伤性白内障组和年龄相关性白内障组患者LECs细胞凋亡率分别为(4.5±2.3)％和(8.7±4.1)％,差异无统计学意义;而糖尿病性白内障组LECs凋亡率为(24.3±6.1)％,与外伤性白内障组及年龄相关性白内障组比较差异有统计学意义(P＜0.05).结论:糖尿病性白内障患者晶体上皮细胞中SIRT1基因及蛋白表达下降,提示该基因参与了糖尿病性白内障的发生,这为我们今后进一步的研究提供了可靠的理论依据.探索调节SIRT1基因在晶状体上皮细胞中表达的有效途径,将为糖尿病性白内障的早期干预治疗提供新的思路.     

Susceptibility of ovine lens crystallins to proteolytic cleavage during formation of hereditary cataract

PURPOSE: To produce two-dimensional electrophoresis (2-DE) maps for ovine crystallins and examine changes in ovine crystallins during cataract formation. METHODS: Soluble and insoluble fractions were isolated from normal, whole lenses of 26-week-old sheep, the proteins separated by 2-DE, and the spots digested with trypsin and subjected to tandem mass spectral analysis. Spot identifications were made by using mass spectrometry data from each spot digestion and data from 2-DE maps of proteins from soluble and insoluble cortices of 10-month-old ovine lens. Ovine alphaA-, alphaB-, and betaB3-crystallin cDNAs were sequenced, whereas other ovine crystallins were identified by using bovine sequences. Proteins were then isolated from whole lenses of 26-week-old lambs with mature hereditary cataracts, and the changes in the crystallins were determined by 2-DE. The masses of truncated crystallins were determined after elution from 2-DE gels. RESULTS: The ovine lens contained the normal complement of crystallins and, similar to other mammalian lenses, underwent partial proteolysis of betaB1-, betaA3-, and betaB3-crystallin during maturation. Cataract development was associated with enhanced truncation of alpha- and beta-crystallins. C-terminal truncations of alphaA- and alphaB-crystallin and N-terminal truncation of betaB2-crystallin were observed as well as a loss of gamma-crystallin. CONCLUSIONS: These data provide the first 2-DE gel maps for ovine lens crystallins and indicated that ovine lens crystallins are truncated during lens maturation. The differences in proteolysis appearing in normal and cataractous lenses suggested that calpain isoforms may be differentially activated during lens maturation and cataract. The ovine hereditary cataract is a useful nonrodent model to study the role of calpain proteolysis in cataract formation.     

三氯乙酸/丙酮蛋白质提取法在白内障晶状体蛋白提取中的应用

目的:比较不同蛋白质提取方法在白内障晶状体蛋白提取中的效果,寻找适合白内障晶状体蛋白质组学研究的蛋白质提取方法。方法:采用裂解液溶解白内障晶状体组织样本,超声裂解离心后分别采用三氯乙酸/丙酮法和ReadyPrep 2D Cleanup Kit对晶状体蛋白样本进行提取、二维电泳,并对电泳后凝胶进行染色、图像采集和图像分析。结果:在2-DE图谱上蛋白斑点的相对分子量在14~97.4kDa之间,等电点在5~9之间,其中高丰度蛋白相对分子量处于20~31kDa范围内,低丰度蛋白斑点相对分子量处于31~43kDa范围内。经TCA/丙酮沉淀法处理的含有透明质酸钠白内障晶状体蛋白样本,二维电泳凝胶图谱背景较清晰,蛋白斑点较清楚,共检测到35~40个左右的蛋白斑点。结论:在人白内障晶状体蛋白质组学研究中,TCA/丙酮法具有较好的纯化效果,为人晶状体组织蛋白质组学的质谱分析研究提供了可靠的二维电泳凝胶图谱。     

Progressive changes in lens crystallin glycation and high-molecular-weight aggregate formation leading to cataract development in streptozotocin-diabetic rats

Because of their remarkable longevity, lens crystallins undergo a substantial amount of glycation (non-enzymatic glycosylation) during diabetic hyperglycemia. These post-translational modifications have the potential to disrupt the structural and functional properties of the lens crystallins and contribute to the formation of cataracts. Streptozotocin-induced diabetic rats were used to study the relationship between glycation of lens proteins and the formation of insoluble high-molecular-weight (HMW) aggregates believed to be responsible for cataract formation. After the onset of diabetes, cataracts developed in about 12- to 13 weeks. The animals were followed in this manner until cataracts developed and for an additional 63 days. Five control and five diabetic rats were killed every 3 weeks and lenses removed. Levels of glycated protein and glycated amino acids in lenses from each animal were examined by affinity chromatography. In addition, the changes in crystallin composition and development of HMW aggregates were monitored by molecular-sieve HPLC techniques. As diabetic hyperglycemia continued there was a linear increase in glycated protein in both the soluble and insoluble fractions. This increase was paralleled by an increase in the soluble HMW and insoluble HMW aggregates. Other changes included a decrease in reactive sulfhydryls which indicates an increase in disulfide bond formation. The gamma-crystallin levels also decreased in a linear fashion during the hyperglycemic pre-cataract and cataract stages. It appears that the glycation of lens crystallin, the disappearance of reactive sulfhydryls and the formation of HMW aggregates are interrelated.     

晶状体蛋白与先天性白内障

先天性白内障是全世界约1/10盲童失明的原因,绝大多数先天性白内障为单基因遗传病,其遗传方式包括常染色体显性遗传、常染色体隐性遗传和X连锁遗传3种。迄今为止在人类及其他动物定位的遗传性先天性白内障的致病基因主要包括编码晶状体结构蛋白、缝隙连接蛋白、膜蛋白、晶状体发育中的调节蛋白的基因。晶状体蛋白是晶状体中最主要的成分,其编码基因的突变与先天性白内障的发生密切相关。了解遗传性先天性白内障的发病机制有助于理解环境及营养因素在晶状体混浊中的作用。就晶状体蛋白的功能、晶状体蛋白基因突变及其导致的先天性白内障表型进行综述。     

Lens proteomics: the accumulation of crystallin modifications in the mouse lens with age.

PURPOSE: To identify modified crystallins associated with aging of lens and produce two-dimensional electrophoresis (2-DE) proteome maps of crystallins in mouse lens. METHODS: Lens proteins from mice of increasing age or different strains were separated by either chromatography or 2-DE. Masses of whole proteins or tryptic peptides were analyzed by mass spectrometry. Changes in the abundance of individual crystallins were determined by image analysis of 2-DE gels. RESULTS: The measured masses of all known mouse crystallins, with the exception of gammaD and gammaF, matched the masses calculated from their reported sequences. Analysis by 2-DE indicated that most posttranslational modifications took place in mice after 6 weeks of age. Partially degraded crystallins, including betaB1, betaB2, betaB3, betaA3, alphaA, and alphaB, were found in greater proportion in the insoluble fractions. gamma-Crystallins A through F also became insoluble during aging. However, insolubilization of gamma-crystallins was associated with a decrease in isoelectric point (pI). Aging was also associated with increased phosphorylation of soluble alphaA- and alphaB-crystallins, confirmed by mass measurements of these proteins eluted from 2-DE gels. Comparison of protein profiles between several strains of mice used to produce transgenic or knockout models of cataract indicated few differences, except for an additional acidic form of a gamma-crystallin, possibly due to a polymorphism. CONCLUSIONS: These results suggest that partial degradation of alpha- and beta-crystallins and increased acidity of gamma-crystallins may cause insolubilization during aging. The 2-DE proteome maps of mouse lens proteins created in this study, using immobilized pH gradients, will be useful for comparison with maps of lens proteins of mice with cataracts so that cataract-specific modifications may be identified.