Quantitative proteomics profiling of sarcomere associated proteins in limb and extraocular muscle allotypes
Quantitative proteomics profiling of sarcomere associated proteins in limb and extraocular muscle allotypes
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DOI：
10.1074/mcp.m600345-mcp200
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发表时间：
2007-04-01
影响因子：
7
通讯作者：
Rubinstein, Neal A.
中科院分区：
文献类型：
--
作者：
Fraterman, Sven;Zeiger, Ulrike;Rubinstein, Neal A.
肌节是横纹肌的主要结构和功能单位。大约有65种不同的蛋白质与肌节相关，它们的确切组成决定了每一块肌肉的速度、耐力和生物学特性。过去的分析严重依赖于电泳和免疫组织化学技术，这些技术一次只能分析一小部分蛋白质。在这里，我们介绍了一种定量的无标记，鸟枪蛋白质组学方法，以快速和可靠的方式，通过液相色谱和质谱法从微克量的肌肉组织中差异定量肌节蛋白。一些肌节蛋白的高序列相似性对鸟枪法蛋白质组学提出了一个问题，因为在随后的数据库搜索算法的限制，在特定亚型的肽的排他性分配。因此，产生了多重序列比对，以改善同种型特异性肽的鉴定。这种方法被用来比较肌节蛋白质组的眼外肌同种异型肢体肌肉。眼外肌是一组独特的高度专业化的肌肉，具有独特的生化，生理和病理特性。我们能够定量40个肌节蛋白;虽然眼外肌中的基本肌节蛋白与肢体肌肉中的那些相似，但稳定Z带与细丝和肋节连接的关键蛋白在眼外肌中增加，并且可能代表对已知通常在眼球运动期间发生的离心收缩的适应。此外，还观察到一些与眼外肌的独特性质密切相关的变化。
The sarcomere is the major structural and functional unit of striated muscle. Approximately 65 different proteins have been associated with the sarcomere, and their exact composition defines the speed, endurance, and biology of each individual muscle. Past analyses relied heavily on electrophoretic and immunohistochemical techniques, which only allow the analysis of a small fraction of proteins at a time. Here we introduce a quantitative label-free, shotgun proteomics approach to differentially quantitate sarcomeric proteins from microgram quantities of muscle tissue in a fast and reliable manner by liquid chromatography and mass spectrometry. The high sequence similarity of some sarcomeric proteins poses a problem for shotgun proteomics because of limitations in subsequent database search algorithms in the exclusive assignment of peptides to specific isoforms. Therefore multiple sequence alignments were generated to improve the identification of isoform specific peptides. This methodology was used to compare the sarcomeric proteome of the extraocular muscle allotype to limb muscle. Extraocular muscles are a unique group of highly specialized muscles with distinct biochemical, physiological, and pathological properties. We were able to quantitate 40 sarcomeric proteins; although the basic sarcomeric proteins in extraocular muscle are similar to those in limb muscle, key proteins stabilizing the connection of the Z-bands to thin filaments and the costamere are augmented in extraocular muscle and may represent an adaptation to the eccentric contractions known to normally occur during eye movements. Furthermore, a number of changes are seen that closely relate to the unique nature of extraocular muscle.