Skeletal muscles are composed of a heterogeneous collection of fiber types with different physiological adaption in response to a stimulus and disease-related conditions. Each fiber has a specific molecular expression of myosin heavy chain molecules (MyHC). So far, MyHCs are currently the best marker proteins for characterization of individual fiber types, and several proteome profiling studies have helped to dissect the molecular signature of whole muscles and individual fibers. Herein, we describe a mass spectrometric workflow to measure skeletal muscle fiber type-specific proteomes. To bypass the limited quantities of protein in single fibers, we developed a Proteomics high-throughput fiber typing (ProFiT) approach enabling profiling of MyHC in single fibers. Aliquots of protein extracts from separated muscle fibers were subjected to capillary LC-MS gradients to profile MyHC isoforms in a 96-well format. Muscle fibers with the same MyHC protein expression were pooled and subjected to proteomic, pulsed-SILAC, and phosphoproteomic analysis. Our fiber type-specific quantitative proteome analysis confirmed the distribution of fiber types in the soleus muscle, substantiates metabolic adaptions in oxidative and glycolytic fibers, and highlighted significant differences between the proteomes of type IIb fibers from different muscle groups, including a differential expression of desmin and actinin-3. A detailed map of the Lys-6 incorporation rates in muscle fibers showed an increased turnover of slow fibers compared to fast fibers. In addition, labeling of mitochondrial respiratory chain complexes revealed a broad range of Lys-6 incorporation rates, depending on the localization of the subunits within distinct complexes. Overall, the ProFiT approach provides a versatile tool to rapidly characterize muscle fibers and obtain fiber-specific proteomes for different muscle groups.

中文翻译：

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高通量蛋白质组学纤维分型（ProFiT），用于全面表征单个骨骼肌纤维。

骨骼肌由对刺激和疾病相关状况具有不同生理适应性的纤维类型的异质集合组成。每条纤维都有肌球蛋白重链分子（MyHC）的特定分子表达。到目前为止，MyHCs目前是表征单个纤维类型的最佳标记蛋白，一些蛋白质组分析研究已经帮助解剖了整个肌肉和单个纤维的分子特征。在这里，我们描述了一种质谱工作流程，用于测量骨骼肌纤维类型特定的蛋白质组。为了绕过单纤维中有限数量的蛋白质，我们开发了Proteomics高通量纤维分型（ProFiT）方法，可对单纤维中的MyHC进行分析。将来自分离的肌肉纤维的蛋白质提取物等分试样进行毛细管LC-MS梯度分析，以96孔形式分析MyHC亚型。汇集具有相同MyHC蛋白表达的肌纤维，并进行蛋白质组学，脉冲SILAC和磷酸化蛋白质组学分析。我们的纤维类型特异性定量蛋白质组学分析证实了比目鱼肌中纤维类型的分布，证实了氧化和糖酵解纤维中的代谢适应，并强调了来自不同肌肉群的IIb型纤维蛋白组之间的显着差异，包括结蛋白的差异表达和actinin-3。肌肉纤维中Lys-6掺入率的详细图谱显示，与快速纤维相比，慢纤维的周转率增加。此外，线粒体呼吸链复合物的标记显示了广泛的Lys-6掺入率，这取决于不同复合物中亚基的定位。总体而言，ProFiT方法提供了一种多功能工具，可快速表征肌肉纤维并获得针对不同肌肉群的特定于纤维的蛋白质组。