Translational Targeted Proteomics Profiling of Mitochondrial Energy Metabolic Pathways in Mouse and Human Samples

Absolute
measurements of protein abundance are important in the
understanding of biological processes and the precise computational
modeling of biological pathways. We developed targeted LC–MS/MS
assays in the selected reaction monitoring (SRM) mode to quantify
over 50 mitochondrial proteins in a single run. The targeted proteins
cover the tricarboxylic acid cycle, fatty acid β-oxidation,
oxidative phosphorylation, and the detoxification of reactive oxygen
species. Assays used isotopically labeled concatemers as internal
standards designed to target murine mitochondrial proteins and their
human orthologues. Most assays were also suitable to quantify the
corresponding protein orthologues in rats. After exclusion of peptides
that did not pass the selection criteria, we arrived at SRM assays
for 55 mouse, 52 human, and 51 rat proteins. These assays were optimized
in isolated mitochondrial fractions from mouse and rat liver and cultured
human fibroblasts and in total liver extracts from mouse, rat, and
human. The developed proteomics approach is suitable for the quantification
of proteins in the mitochondrial energy metabolic pathways in mice,
rats, and humans as a basis for translational research. Initial data
show that the assays have great potential for elucidating the adaptive
response of human patients to mutations in mitochondrial proteins
in a clinical setting.