Di Antonio Chiara, Marabelli Chiara, Bongianino Rossana, Priori Silvia G
Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy.
Laboratory of Molecular Cardiology, IRCCS ICS Maugeri, 27100 Pavia, Italy.
Int J Mol Sci. 2025 Sep 4;26(17):8602. doi: 10.3390/ijms26178602.
The junctional sarcoplasmic reticulum (jSR) is a critical organelle in cardiomyocytes, regulating calcium homeostasis and Excitation-Contraction Coupling (ECC). A quantitative understanding of its protein composition is essential for investigating cardiac physiology and related pathologies. However, isolating intact jSR vesicles, particularly those enriched in membrane proteins, remains a challenging task. Here, we describe our optimized protocol for reproducible enrichment of jSR vesicles from a single murine heart, without the use of antibodies. The protocol enables the recovery of low-abundance membrane proteins while preserving their native interactions with partners. This strategy facilitates the straightforward identification by Mass Spectrometry of highly relevant yet challenging jSR proteins, including the cardiac Ryanodine Receptor and calsequestrin. Our protocol provides a robust tool for studying the structural and stoichiometric organization of the cardiac jSR components in a widely used animal model.
连接肌浆网（jSR）是心肌细胞中的一种关键细胞器，调节钙稳态和兴奋-收缩偶联（ECC）。对其蛋白质组成进行定量了解对于研究心脏生理学和相关病理学至关重要。然而，分离完整的jSR囊泡，尤其是富含膜蛋白的囊泡，仍然是一项具有挑战性的任务。在这里，我们描述了我们优化的方案，用于从单个小鼠心脏中可重复富集jSR囊泡，且不使用抗体。该方案能够回收低丰度膜蛋白，同时保留它们与伙伴的天然相互作用。这种策略有助于通过质谱直接鉴定高度相关但具有挑战性的jSR蛋白，包括心脏雷诺丁受体和肌集钙蛋白。我们的方案为在广泛使用的动物模型中研究心脏jSR组件的结构和化学计量组织提供了一个强大的工具。
