Departments of Physiology and Medicine, Division of Cardiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
J Proteomics. 2012 Aug 3;75(15):4602-9. doi: 10.1016/j.jprot.2012.02.014. Epub 2012 Feb 22.
The regulation of mitochondrial function is essential for cardiomyocyte adaptation to cellular stress. While it has long been understood that phosphorylation regulates flux through metabolic pathways, novel phosphorylation sites are continually being discovered in all functionally distinct areas of the mitochondrial proteome. Extracting biologically meaningful information from these phosphorylation sites requires an adaptable, sensitive, specific and robust method for their quantification. Here we report a multiple reaction monitoring-based mass spectrometric workflow for quantifying site-specific phosphorylation of mitochondrial proteins. Specifically, chromatographic and mass spectrometric conditions for 68 transitions derived from 23 murine and human phosphopeptides, and their corresponding unmodified peptides, were optimized. These methods enabled the quantification of endogenous phosphopeptides from the outer mitochondrial membrane protein VDAC, and the inner membrane proteins ANT and ETC complexes I, III and V. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of mitochondrial protein phosphorylation in cardiac physiology and pathophysiology. This article is part of a Special Issue entitled: Translational Proteomics.
线粒体功能的调节对于心肌细胞适应细胞应激至关重要。虽然人们早就知道磷酸化调节代谢途径中的通量，但在线粒体蛋白质组的所有功能不同的区域中，新的磷酸化位点仍在不断被发现。从这些磷酸化位点中提取有生物学意义的信息需要一种适应性强、灵敏、特异和稳健的定量方法。在这里，我们报告了一种基于多重反应监测的质谱工作流程，用于定量线粒体蛋白的特异性磷酸化。具体来说，优化了 23 个鼠和人磷酸肽及其相应未修饰肽的 68 个跃迁的色谱和质谱条件。这些方法使内、外线粒体膜蛋白 VDAC、ANT 和 ETC 复合物 I、III 和 V 的内源性磷酸肽的定量成为可能。该定量工作流程的开发是推进我们对心肌生理学和病理生理学中线粒体蛋白磷酸化的调节作用的认识和理解的关键步骤。本文是一个题为“转化蛋白质组学”的特刊的一部分。
