Metabolome and Proteome Profiling of Complex I Deficiency Induced by Rotenone

Abstract

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.

Ataxin-2 (Atxn2)-Knock-Out Mice Show Branched Chain Amino Acids and Fatty Acids Pathway Alterations

“…The correct retention time, three MRM's and a matching MRM ion ratio of tuned pure metabolites as a reference were used for correct identification of metabolites (44). In total, 176 metabolites could be identified and relatively quantified in liver tissue, 204 in cerebellum.…”

“…MS settings were reported previously (44), transition settings for the MRM's in supplemental Table S2.…”

“…Metabolome Data Analysis-Relative quantification was performed using MultiQuant software v.2.1.1 (AB Sciex, Foster City, CA), as described previously (44). Briefly, the identification of metabolites was based on the retention time, three co-occurring transitions and the ion ratio between the transitions, which had to match to MRM ion ratios of the corresponding tuned metabolites.…”

“…Lysates were sonicated on ice for 1 min and boiled at 95°C for 5 min at a rocking platform (750 rpm). Samples were prepared for LC-MS analysis as previously (44). In brief, lysates were centrifuged for 5 min at 15,000 ϫ g, supernatants were reduced in 50 mM DTT for 1 h at 56°C.…”

An Impaired Respiratory Electron Chain Triggers Down-regulation of the Energy Metabolism and De-ubiquitination of Solute Carrier Amino Acid Transporters

“…For quantitative proteome profiling, a shotgun LC-MS/MS approach including the classical SILAC labeling was performed. For comprehensive metabolome profiling, a targeted LC-MS approach, based on multiple reaction monitoring (MRM) (19) was applied.…”