Proteomic analysis of exfoliation deposits
Proteomic analysis of exfoliation deposits
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DOI：
10.1167/iovs.06-0411
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发表时间：
2007-04-01
影响因子：
4.4
通讯作者：
Ritch, Robert
中科院分区：
文献类型：
--
作者：
Ovodenko, Boris;Rostagno, Agueda;Ritch, Robert
目的。目的：通过蛋白质组学方法增加对晶状体剥脱材料(XFM)生化成分的认识。方法：将有剥落综合征(XFS)和无剥脱综合征(XFS)患者的晶状体前囊在甲酸中匀浆，并用溴化氰(CN-BR)裂解，银染后用SDS-PAGE比较化学产生的碎片的模式。切除对照病例中不存在的独特XFS条带，用TPCK-胰酶消化，并用四极飞行时间质谱仪(MS)对所得多肽进行测序。在平行实验中，用胰酶和弹性酶分别在溶液中消化CNBr片段的XFM，得到的多肽混合物用串联MS进行分析，然后通过在非冗余蛋白质数据库中进行同源搜索来鉴定。结果除了已知的XFM细胞外基质和基底膜成分纤维蛋白-1、纤维连接蛋白、玻璃体连接蛋白、层粘连蛋白和淀粉样蛋白P组分外，蛋白质组学方法还鉴定了多功能蛋白质簇蛋白和金属蛋白酶组织抑制因子(TIMP)-3以及新的分子，其中包括纤毛蛋白-2、纤维粘连蛋白-2、糖胺多聚糖合成酶-3，以及ADAM家族的粘附素、金属膜蛋白和经典补体激活途径C1q的起始成分。结论一种新的增溶策略结合敏感的蛋白质分析强调了XFS沉积的复杂性，并为研究XFS的发生和发展的分子机制开辟了新的途径。
PURPOSE. To increase knowledge of the biochemical composition of lenticular exfoliation material (XFM) by using proteomic approaches.METHODS. Anterior lens capsules from patients with and without exfoliation syndrome (XFS) were homogenized in formic acid and subjected to cyanogen bromide (CN-Br) cleavage, and the pattern of chemically generated fragments was compared by SDS-PAGE after silver staining. Unique XFS bands not present in control cases were excised, digested with TPCK-trypsin, and the resultant peptides sequenced with quadrupole time-of-flight mass spectrometry (MS). In parallel experiments, CNBr-fragmented XFM was separately digested in solution with trypsin and elastase, and the resultant peptide mixture was analyzed by liquid chromatography coupled to tandem MS followed by identification through homology searches at non-redundant protein databases. Immunolocalization of the MS-identified components were performed in XFS versus control samples by using conventional immumohistochemical methods and light microscopy.RESULTS. In addition to fibrillin-1, fibronectin, vitronectin, laminin, and amyloid P-component, which are well-known extracellular matrix and basement membrane components of XFM, the proteornic approaches identified the multifunctional protein clusterin and tissue inhibitor of metalloprotease (TIMP)-3 as well as novel molecules, among them fibulin-2, desmocollin-2, the glycosaminoglycans syndecan-3, and versican, membrane metalloproteases of the ADAM family (a disintegrin and metalloprotease), and the initiation component of the classic complement activation pathway C1q. In all cases, classic imnumohistochemistry confirmed their location in XFM.CONCLUSIONS. A novel solubilization strategy combined with sensitive proteornic analysis emphasizes the complexity of the XFS deposits and opens new avenues to study the molecular mechanisms involved in the pathogenesis and progression of XFS.