A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics.
A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics.
复制标题
高通量人类单骨骼肌纤维蛋白质组学的完整工作流程。
DOI：
10.1021/jasms.3c00072
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发表时间：
2023
影响因子：
3.2
通讯作者：
Meyer,JesseG
中科院分区：
文献类型：
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作者：
Momenzadeh,Amanda;Jiang,Yuming;Kreimer,Simion;Teigen,LauraE;Zepeda,CarlosS;Haghani,Ali;Mastali,Mitra;Song,Yang;Hutton,Alexandre;Parker,SarahJ;VanEyk,JenniferE;Sundberg,ChristopherW;Meyer,JesseG
骨骼肌是全身代谢的主要调节组织，由多种细胞（纤维）类型的混合物组成。衰老和几种疾病对各种纤维类型的影响不同，因此，以纤维类型特定的方式研究蛋白质组的变化是必要的。最近在分离的单个肌肉纤维蛋白质组学方面的突破已经开始揭示纤维之间的异质性。然而，现有的方法缓慢且费力，每根肌肉纤维需要2小时的质谱分析时间；50根纤维大约需要4天的时间来分析。因此，要捕获个体内部和个体之间纤维的高变异性，需要在高通量单肌肉纤维蛋白质组学方面取得进展。在这里，我们使用单细胞蛋白质组学方法在15分钟的总仪器时间内对单个肌纤维蛋白质组进行定量。作为概念证明，我们在13.25小时内分析了来自两个健康个体的53个分离骨骼肌纤维的数据。采用单细胞数据分析技术来整合数据，我们可以可靠地分离出1型和2A型纤维。94种蛋白质在簇之间有统计学差异，表明参与脂肪酸氧化、氧化磷酸化、肌肉结构和收缩功能的蛋白质发生了改变。我们的结果表明，该方法在保持足够的蛋白质组深度的同时，在数据收集和样品制备方面都明显快于先前的单纤维方法。我们预计该分析将使未来研究跨越数百个人的单个肌肉纤维成为可能，这在以前由于吞吐量的限制是不可能的。
Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious, requiring 2 h of mass spectrometry time per single muscle fiber; 50 fibers would take approximately 4 days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 min total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 h. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Ninety-four proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, oxidative phosphorylation, and muscle structure and contractile function. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput.