de Souza Gustavo A, Godoy Lyris M F, Mann Matthias
Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej, Odense M, Denmark.
Genome Biol. 2006;7(8):R72. doi: 10.1186/gb-2006-7-8-R72. Epub 2006 Aug 10.
The tear film is a thin layer of fluid that covers the ocular surface and is involved in lubrication and protection of the eye. Little is known about the protein composition of tear fluid but its deregulation is associated with disease states, such as diabetic dry eyes. This makes this body fluid an interesting candidate for in-depth proteomic analysis.
In this study, we employ state-of-the-art mass spectrometric identification, using both a hybrid linear ion trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) mass spectrometer, and high confidence identification by two consecutive stages of peptide fragmentation (MS/MS/MS or MS3), to characterize the protein content of the tear fluid. Low microliter amounts of tear fluid samples were either pre-fractionated with one-dimensional SDS-PAGE and digested in situ with trypsin, or digested in solution. Five times more proteins were detected after gel electrophoresis compared to in solution digestion (320 versus 63 proteins). Ontology classification revealed that 64 of the identified proteins are proteases or protease inhibitors. Of these, only 24 have previously been described as components of the tear fluid. We also identified 18 anti-oxidant enzymes, which protect the eye from harmful consequences of its exposure to oxygen. Only two proteins with this activity have been previously described in the literature.
Interplay between proteases and protease inhibitors, and between oxidative reactions, is an important feature of the ocular environment. Identification of a large set of proteins participating in these reactions may allow discovery of molecular markers of disease conditions of the eye.
泪膜是覆盖眼表的一层薄液层，参与眼的润滑和保护。关于泪液的蛋白质组成知之甚少，但其失调与疾病状态相关，如糖尿病性干眼。这使得这种体液成为深入蛋白质组学分析的一个有趣候选对象。
在本研究中，我们采用了最先进的质谱鉴定方法，使用混合线性离子阱 - 傅里叶变换（LTQ - FT）和线性离子阱 - 轨道阱（LTQ - Orbitrap）质谱仪，并通过肽段连续两级碎裂（MS/MS/MS或MS3）进行高可信度鉴定，以表征泪液的蛋白质含量。低微升量的泪液样本要么用一维SDS - PAGE预分级并原位用胰蛋白酶消化，要么在溶液中消化。与溶液消化相比，凝胶电泳后检测到的蛋白质多五倍（320种对63种）。本体分类显示，鉴定出的64种蛋白质是蛋白酶或蛋白酶抑制剂。其中，只有24种先前被描述为泪液成分。我们还鉴定出18种抗氧化酶，它们保护眼睛免受暴露于氧气的有害后果。文献中先前仅描述了两种具有这种活性的蛋白质。
蛋白酶与蛋白酶抑制剂之间以及氧化反应之间的相互作用是眼部环境的一个重要特征。鉴定参与这些反应的大量蛋白质可能有助于发现眼部疾病状况的分子标志物。
