Kallabis Sebastian, Abraham Lena, Müller Stefan, Dzialas Verena, Türk Clara, Wiederstein Janica Lea, Bock Theresa, Nolte Hendrik, Nogara Leonardo, Blaauw Bert, Braun Thomas, Krüger Marcus
CECAD Research Center, Institute for Genetics, University of Cologne, 50931, Cologne, Germany.
Max Planck Institute for the Biology of Aging, 50931, Cologne, Germany.
Skelet Muscle. 2020 Mar 23;10(1):7. doi: 10.1186/s13395-020-00226-5.
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.
骨骼肌由多种不同类型的纤维组成，这些纤维在对刺激和疾病相关状况的生理适应方面存在差异。每根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，MyHC是目前用于表征个体纤维类型的最佳标记蛋白，多项蛋白质组分析研究有助于剖析整个肌肉和单个纤维的分子特征。
在此，我们描述了一种用于测量骨骼肌纤维类型特异性蛋白质组的质谱工作流程。为了克服单根纤维中蛋白质数量有限的问题，我们开发了一种蛋白质组学高通量纤维分型（ProFiT）方法，能够对单根纤维中的MyHC进行分析。从分离的肌肉纤维中提取的蛋白质等分试样进行毛细管液相色谱 - 质谱梯度分析，以96孔板形式分析MyHC异构体。将具有相同MyHC蛋白表达的肌肉纤维汇集起来，进行蛋白质组学、脉冲式稳定同位素标记氨基酸法（pulsed-SILAC）和磷酸化蛋白质组分析。
我们的纤维类型特异性定量蛋白质组分析证实了比目鱼肌中纤维类型的分布，证实了氧化纤维和糖酵解纤维中的代谢适应性，并突出了来自不同肌肉群的IIb型纤维蛋白质组之间的显著差异，包括结蛋白和辅肌动蛋白-3的差异表达。肌肉纤维中赖氨酸-6掺入率的详细图谱显示，慢纤维的周转率比快纤维高。此外，线粒体呼吸链复合物的标记显示，根据亚基在不同复合物中的定位，赖氨酸-6掺入率范围广泛。
总体而言，ProFiT方法提供了一种通用工具，可快速表征肌肉纤维并获得不同肌肉群的纤维特异性蛋白质组。
