Tissue Specific Phosphorylation of Mitochondrial Proteins Isolated from Rat Liver, Heart Muscle, and Skeletal Muscle

Phosphorylation of mitochondrial
proteins in a variety of biological
processes is increasingly being recognized and may contribute to the
differences in function and energy demands observed in mitochondria
from different tissues such as liver, heart, and skeletal muscle.
Here, we used a combination of TiO2 phosphopeptide-enrichment,
HILIC fractionation, and LC–MS/MS on isolated mitochondria
to investigate the tissue-specific mitochondrial phosphoproteomes
of rat liver, heart, and skeletal muscle. In total, we identified
899 phosphorylation sites in 354 different mitochondrial proteins
including 479 potential novel sites. Most phosphorylation sites were
detected in liver mitochondria (594), followed by heart (448) and
skeletal muscle (336), and more phosphorylation sites were exclusively
identified in liver mitochondria than in heart and skeletal muscle.
Bioinformatics analysis pointed out enrichment for phosphoproteins
involved in amino acid and fatty acid metabolism in liver mitochondria,
whereas heart and skeletal muscle were enriched for phosphoproteins
involved in energy metabolism, in particular, tricarboxylic acid cycle
and oxidative phosphorylation. Multiple tissue-specific phosphorylation
sites were identified in tissue-specific enzymes such as those encoded
by HMGCS2, BDH1, PCK2, CPS1, and OTC in liver mitochondria,
and CKMT2 and CPT1B in heart and
skeletal muscle. Kinase prediction showed an important role for PKA
and PKC in all tissues but also for proline-directed kinases in liver
mitochondria. In conclusion, we provide a comprehensive map of mitochondrial
phosphorylation sites, which covers approximately one-third of the
mitochondrial proteome and can be targeted for the investigation of
tissue-specific regulation of mitochondrial biological processes.