Quantitative mitochondrial phosphoproteomics using iTRAQ on an LTQ-Orbitrap with high energy collision dissociation.
With the use of iTRAQ labeling and mass spectrometry on an LTQ-Orbitrap with HCD capability, we assessed relative changes in protein phosphorylation in the mitochondria upon physiological perturbation. As a reference reaction, we monitored the well-characterized regulation of pyruvate dehydrogenase (PDH) activity via phosphorylation/dephosphorylation by pyruvate dehydrogenase kinase/pyruvate dehydrogenase phosphatase in response to dichloroacetate, de-energization and Ca2+. Relative quantification of phosphopeptides of PDH-E1alpha subunit from porcine heart revealed dephosphorylation at three serine sites (Ser231, Ser292 and Ser299). Dephosphorylation at Ser292 (i.e., the inhibitory site) with DCA correlated with an activation of PDH activity as previously reported, consistent with our de-energization data. Calcium also dephosphorylated (i.e., activated) PDH, thus, confirming calcium activation of PDP. With this approach, we successfully monitored other phosphorylation sites of mitochondrial proteins including adenine nucleotide translocase, malate dehydrogenase and mitochondrial creatine kinase. Among them, four proteins exhibited phosphorylation changes with these physiological stimuli: (1) BCKDH-E1alpha subunit increased phosphorylation at Ser337 with DCA and de-energization; (2) apoptosis-inducing factor phosphorylation was elevated at Ser345 with calcium; (3) ATP synthase F1 complex alpha subunit and (4) mitofilin dephosphorylated at Ser65 and Ser264 upon de-energization. This screening validated the iTRAQ/HCD technology as a method for functional quantitation of mitochondrial protein phosphorylation as well as providing insight into the regulation of mitochondria via phosphorylation.
Boja
Emily S
ES
Proteomics Core Facility, National Heart, Lung and Blood Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892-1061, USA.
Phillips
Darci
D
French
Stephanie A
SA
Harris
Robert A
RA
Balaban
Robert S
RS
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003999:Dichloroacetic Acid / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D008929:Mitochondria, Heart / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000032:analysis* / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase / Q000378:metabolism; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D013552:Swine
D024101:Mitochondrial Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D003999:Dichloroacetic Acid; D017346:Protein Serine-Threonine Kinases; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase; D002118:Calcium
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