Phosphoproteome analysis reveals regulatory sites in major pathways of cardiac mitochondria

Abstract

Mitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation, including components of the electron transport chain (ETC) complexes and enzymes involved in metabolic pathways (e.g. tricarboxylic acid cycle). Furthermore, calcium overload injured cardiac mitochondrial ETC function, whereas enhanced phosphorylation of ETC via application of phosphatase inhibitors restored calcium-attenuated ETC complex I and complex III activities, demonstrating positive regulation of ETC function by phosphorylation. Moreover, in silico analyses of the identified phosphopeptide motifs illuminated the molecular nature of participating kinases, which included several known mitochondrial kinases (e.g. pyruvate dehydrogenase kinase) as well as kinases whose mitochondrial location was not previously appreciated (e.g. Src). In conclusion, the phosphorylation events defined herein advance our understanding of cardiac mitochondrial biology, facilitating the integration of the still fragmentary knowledge about mitochondrial signaling networks, metabolic pathways, and intrinsic mechanisms of functional regulation in the heart.

Fig. 1.

Identification of novel phosphorylation events in diverse pathways of cardiac mitochondria. A, mass spectrum for ubiquinol-cytochrome c reductase core protein 1, a subunit of complex III in the mitochondrial electron transport chain. The peptide was phosphorylated at serine residue 212 (Sp) as detected by ETD analyses. B, creatine kinase, which is an essential energy metabolism protein in mitochondria, was shown by CID analysis to be phosphorylated at serine 319. A strong neutral loss signal was observed in the MS2 spectrum (top). The peptide precursors with phosphate neutral loss were isolated and further characterized by MS3 (bottom). C, oxoglutarate dehydrogenase, an important component of the mitochondrial tricarboxylic acid cycle, was identified by both ETD (top) and CID (bottom) to be phosphorylated at threonine residue 502.

Fig. 2.

Phosphorylation plays essential role in mitochondrial complex I and III activity down-regulation induced by calcium loading. Mitochondrial complex I (A) and III (B) activities were suppressed by calcium treatment (40 μm). The calcium-sensitive inhibition of complex I activity was reversed independently with three phosphatase inhibitors, fenvalerate, calyculin A, and okadaic acid. The calcium-sensitive inhibition of complex III was reversed by fenvalerate, whereas calyculin A and okadaic acid showed marginal effects. *, p < 0.05 versus control + vehicle group; #, p < 0.05 versus calcium + vehicle group; n = 6 per group. The error bars represent standard deviation.

Fig. 3.

Working scheme to delineate phosphorylation events in diverse mitochondrial pathways. A, many of these regulatory sites are involved in the major mitochondrial pathways of metabolism and transport. By combining both ETD and CID technologies, it was possible to ascertain a total of 18 and 10 phosphorylation sites from the TCA cycle and ETC complexes, respectively. A further 12, two, and one phosphorylation sites were also identified for fatty acid metabolism, creatine kinase, and the urea cycle, respectively. A number of additional phosphorylation sites were identified in ANT (5), nicotinamide nucleotide transhydrogenase (NNT) (1), and voltage-dependent anion channel (VDAC) (1), which are major regulatory proteins involved in transport. The identified phosphoproteins involved in the ETC complexes and TCA cycle are illustrated in B and C, respectively. The identified phosphoproteins and the number of identified phosphorylation sites are shown. The number showing in parenthesis represents the number of phosphorylation sites identified for the listed protein. mtCK, mitochondrial creatine kinase.

Fig. 4.

Characterization of murine mitochondrial phosphoproteome utilizing two types of fragmentation, namely ETD and CID. A, plot showing the correlation between the confidence level of ETD-based identifications and the E-value obtained from OMSSA. E-values were expressed to log₁₀. The confidence level was examined by searching all the spectra against a sequence-reversed decoy database. B, plot examining the correlation between the confidence of CID-based identifications and the Xcorr values from three distinct charge states (z = 1+, z = 2+, and z = 3+). The confidence level was scrutinized in a similar way to the ETD analyses using a decoy database. C, the accumulated numbers of identified phosphopeptides and phosphorylation sites from the ETD analyses plotted against our number of biological replicates. No significant increases in identifications of phosphopeptides and phosphorylation sites were observed after using four biological replicates. D, the accumulated number of identifications plotted against the number of biological replicates for the CID analyses. No significant increases in identifications were observed after using five biological replicates. E, pie chart illustrating the complementary identifications of the mitochondrial phosphorylation sites by both ETD and CID. Each fragmentation process has a distinct preference in peptide sequencing, underlining the importance of our combined approach to increase the identification of mitochondrial phosphorylation sites. F, graph emphasizing the significant expansion in the mitochondrial phosphoproteome data set as identified in this study. 210 phosphorylation sites and 82 phosphoproteins were novel identifications.

Fig. 5.

Performance characteristics of ETD/CID in this investigation. A, pie chart depicting the percentage of our identified phosphopeptides according to different charge states for the ETD analysis. ETD performed better for a 3+ charge state. B, pie chart depicting the percentage of the identified phosphopeptides according to different charge states for the CID analysis. As expected, CID performed better for doubly charged precursors. C, bar chart depicting the number of identified phosphopeptides as a function of precursor m/z for the ETD analysis. As can be seen, ETD performed better for precursors with an m/z of 600 or less. D, bar chart depicting the number of identified phosphopeptides as a function of precursor m/z for the CID analysis. As can be seen, CID performed better for precursors with an m/z of 600 or more. E, scatter plot comparing the distribution of precursor m/z of the identified phosphopeptides by both ETD and CID analyses.

Fig. 6.

Functional annotation of identified mitochondrial phosphoproteins. A, pie chart showing the functional annotation of the 181 identified phosphoproteins based on their protein functions. Of the 181 annotated phosphoproteins, 38 were involved in critical metabolic mechanisms that are essential for healthy cardiac mitochondria, including the TCA cycle, fatty acid transport/oxidation, and nucleic acid metabolism. B, the identified phosphoproteins were annotated for both their cell locations (horizontal) and their protein function (vertical) in the form of a heat map. The number of mitochondrial phosphoproteins within each functional category is listed in parentheses. ER, endoplasmic reticulum.