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.

Figure 1. General Workflow for the MRM-based Quantification of Cardiac Mitochondrial Protein Phosphorylation

1) Synthetic peptides labeled with heavy isotopes are subjected to LC-MS/MS to determine potential transitions. A, Choosing MRM transitions from the MS/MS spectrum of the heavy-labeled VLDsGAPIKIPVGPETLGR phosphopeptide standard. B, Traces for three transitions from the same peptide during MRM optimization of fragmentor voltage and collision energy. C, LC/MS/MS-MRM (21 total transitions) total ion current display following injection of a mitochondrial digest showing quality of LC separation, with the arrow indicating the peak for the VLDsGAPIKIPVGPETLGR peptides. 2) Murine cardiac mitochondria are isolated. 3) Coomassie stained BN-PAGE of the mitochondrial protein preparation shows the separation of Complexes I, III and V. 4) MRM traces for quantification of the heavy standard and light endogenous forms of the VLDsGAPIKIPVGPETLGR peptide.

Figure 2. Optimization of Collision Energy Using Optimizer for Peptides and Application of Optimized MRM Parameters to a Mitochondrial Sample

A) The collision energy (CE) for each transition was optimized by increasing the CE at an interval of 4V (from 0V to 50V) while acquiring in MRM mode. The CE giving rise to the most intense peak area was selected by manual inspection. B) Plot of peak area versus CE voltage for the m/z 858.5→988.5 transition. C) Representative overlaid LC-MRM chromatograms (3 transitions per peptide) illustrating chromatographic alignment and symmetry of the selected transitions (refer to Supp. Figure S1 for non-overlaid chromatograms). Peaks are labeled with peptide ID (refer to Table 1). P: phosphorylated peptide; N: unmodified peptide). D) LC-MRM signals from phosphopeptides, and E) unmodified peptides detected in the endogenous mitochondrial sample. Heavy standard peptide signals are shown above the corresponding light endogenous peptide signals. Transitions are displayed in overlaid mode for each peptide and m/z values are labeled.