Highly sensitive saturation labeling reveals changes in abundance of cell cycle-associated proteins and redox enzyme variants during oocyte maturation in vitro
Highly sensitive saturation labeling reveals changes in abundance of cell cycle-associated proteins and redox enzyme variants during oocyte maturation in vitro
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DOI：
10.1002/pmic.200700417
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发表时间：
2009-02-01
期刊：
影响因子：
3.4
通讯作者：
Arnold, Georg J.
中科院分区：
文献类型：
--
作者：
Berendt, Frank J.;Froehlich, Thomas;Arnold, Georg J.
卵母细胞成熟是一个复杂的过程，也是人类和其他哺乳动物辅助生殖技术（ART）中的关键问题。我们使用了一个敏感的2-D DIGE饱和标记的方法，包括一个内部合并标准的定量蛋白质组分析的未成熟与体外成熟的牛卵母细胞在6个独立的样品。该研究包括代表24个DIGE实验的48个2D凝胶图像。从每个凝胶分析的250 ng样品中，定量分析显示pH 4-7图像中平均2244个斑点，pH 6-9图像中平均1291个斑点。共检测到38个不同强度的斑点。通过nano-LC-MS/MS分析鉴定来自2200个卵母细胞的制备凝胶的斑点。可以明确鉴定的10个点包括Ca 2+结合蛋白质、抑制性控制的肿瘤蛋白质、克雷布斯和戊糖磷酸循环的酶、丛生蛋白、14-3-3 β、延伸因子-1 γ和氧化还原酶如GST Mu 5和过氧化物酶氧还蛋白-3的多态形式。通过激光共聚焦扫描显微镜测定两种蛋白的细胞分布。这项研究确定的有趣的候选蛋白可能有助于改善体外成熟过程，以提高牛和其他哺乳动物体外受精和其他ART的成功率。
Oocyte maturation is a complex process and a critical issue in assisted reproduction techniques (ART) in humans and other mammals. We used a sensitive 2-D DIGE saturation labeling approach including an internal pooled standard for quantitative proteome profiling of immature versus in vitro matured bovine oocytes in six independent samples. The study comprised 48 2D gel images representing 24 DIGE experiments. From 250 ng sample analyzed per gel, quantitative analysis revealed an average of 2244 spots in pH 4-7 images and 1291 spots in pH 6-9 images. Thirty-eight spots with different intensities were detected in total. Spots of a preparative gel from 2200 oocytes were identified by nano-LC-MS/MS analysis. The ten spots which could be unambiguously identified include the Ca2+-binding protein translationally controlled tumor protein, enzymes of the Krebs and pentose phosphate cycles, clusterin, 14-3-3 epsilon, elongation factor-1 gamma, and redox enzymes such as polymorphic forms of GST Mu 5 and peroxiredoxin-3. The cellular distribution of two proteins was determined by confocal laser scanning microscopy. The interesting protein candidates identified by this study may help to improve the in vitro maturation process in order to increase the rate of successful in vitro fertilization and other ART in cattle and other mammals.