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.

多种生理过程中线粒体蛋白的磷酸化正愈发受到学界重视，其或与肝、心、骨骼肌等不同组织来源的线粒体在功能及能量需求上的差异密切相关。
本研究结合二氧化钛（TiO₂）磷酸肽富集、亲水相互作用色谱（HILIC）分级分离及液相色谱-串联质谱（LC–MS/MS）技术，对分离得到的线粒体进行分析，以探究大鼠肝、心及骨骼肌的组织特异性线粒体磷酸化蛋白质组。
本研究共在354种不同线粒体蛋白中鉴定出899个磷酸化位点，其中包含479个潜在新型磷酸化位点。其中，肝线粒体中检测到的磷酸化位点最多（594个），其次为心脏（448个）与骨骼肌（336个）；仅在肝线粒体中鉴定到的磷酸化位点数量多于心脏与骨骼肌。
生物信息学分析显示，肝线粒体中的磷酸化蛋白显著富集于氨基酸与脂肪酸代谢通路；而心脏与骨骼肌的磷酸化蛋白则富集于能量代谢通路，尤其是三羧酸循环与氧化磷酸化过程。
研究还在组织特异性酶中鉴定出多个组织特异性磷酸化位点：肝线粒体中的相关酶由HMGCS2、BDH1、PCK2、CPS1及OTC编码，而心脏与骨骼肌中的相关酶则由CKMT2与CPT1B编码。
激酶预测分析表明，蛋白激酶A（PKA）与蛋白激酶C（PKC）在所有组织的线粒体磷酸化过程中均发挥重要作用，而肝线粒体中脯氨酸定向激酶也扮演了关键角色。
综上，本研究构建了一套全面的线粒体磷酸化位点图谱，该图谱覆盖了约三分之一的线粒体蛋白质组，可用于后续探究线粒体生理过程的组织特异性调控机制。