title: An MRM-based workflow for quantifying cardiac mitochondrial protein phosphorylation in murine and human tissue.
abstract: 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.
authors.affiliation: Departments of Physiology and Medicine, Division of Cardiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
authors.forename: Maggie P Y
authors.initials: MP
authors.lastname: Lam
authors.forename: Sarah B
authors.initials: SB
authors.lastname: Scruggs
authors.forename: Tae-Young
authors.initials: TY
authors.lastname: Kim
authors.forename: Chenggong
authors.initials: C
authors.lastname: Zong
authors.forename: Edward
authors.initials: E
authors.lastname: Lau
authors.forename: Ding
authors.initials: D
authors.lastname: Wang
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Ryan
authors.forename: Kym F
authors.initials: KF
authors.lastname: Faull
authors.forename: Peipei
authors.initials: P
authors.lastname: Ping
mesh_terms: D000818:Animals; D006801:Humans; D051379:Mice; D008929:Mitochondria, Heart / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*
chemical_list: D024101:Mitochondrial Proteins; D009124:Muscle Proteins