Metabolome and Proteome Profiling of Complex I Deficiency Induced by Rotenone

Complex
I (CI; NADH dehydrogenase) deficiency causes mitochondrial
diseases, including Leigh syndrome. A variety of clinical symptoms
of CI deficiency are known, including neurodegeneration. Here, we
report an integrative study combining liquid chromatography–mass
spectrometry (LC–MS)-based metabolome and proteome profiling
in CI deficient HeLa cells. We report a rapid LC–MS-based method
for the relative quantification of targeted metabolome profiling with
an additional layer of confidence by applying multiple reaction monitoring
(MRM) ion ratios for further identity confirmation and robustness.
The proteome was analyzed by label-free quantification (LFQ). More
than 6000 protein groups were identified. Pathway and network analyses
revealed that the respiratory chain was highly deregulated, with metabolites
such as FMN, FAD, NAD+, and ADP, direct players of the
OXPHOS system, and metabolites of the TCA cycle decreased up to 100-fold.
Synthesis of functional iron–sulfur clusters, which are of
central importance for the electron transfer chain, and degradation
products like bilirubin were also significantly reduced. Glutathione
metabolism on the pathway level, as well as individual metabolite
components such as NADPH, glutathione (GSH), and oxidized glutathione
(GSSG), was downregulated. Overall, metabolome and proteome profiles
in CI deficient cells correlated well, supporting our integrated approach.