An MRM-based workflow for quantifying cardiac mitochondrial protein phosphorylation in murine and human tissue

• Published in: Journal of proteomics Vol. 75; no. 15; pp. 4602 - 4609
• Main Authors: Lam, Maggie P.Y., Scruggs, Sarah B., Kim, Tae-Young, Zong, Chenggong, Lau, Edward, Wang, Ding, Ryan, Christopher M., Faull, Kym F., Ping, Peipei
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 03.08.2012
• Subjects: Animals; biochemical pathways; cardiomyocytes; Cardiovascular disease; chromatography; Humans; membrane proteins; Mice; Mitochondria; Mitochondria, Heart - metabolism; mitochondrial membrane; Mitochondrial Proteins - metabolism; MRM; Muscle Proteins - metabolism; Myocardium - metabolism; pathophysiology; peptides; Phosphorylation; physiology; protein phosphorylation; Protein quantitation; proteome; proteomics; Proteomics - methods; Protein quantitation; Cardiovascular disease; MRM; Phosphorylation; Mitochondria
• ISSN: 1874-3919; 1876-7737
• DOI: 10.1016/j.jprot.2012.02.014

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. [Display omitted] ► Studying mitochondrial phosphorylation requires sensitive and reliable quantitative methods. ► A workflow combining peptide separation and multiple reaction monitoring is described. ► Optimization of 68 transitions from phosphorylation sites in mouse and human ► Quantification of 11 endogenous peptides using the optimized MRM method