Fig. 1.

Strategy for identification and quantitation of the SIRT3-regulated acetylome in mouse liver mitochondria. Liver mitochondria were isolated from five individual WT and SIRT3^−/− male mice. Two process replicates from each of 10 mitochondrial protein isolates were digested separately with trypsin, generating 20 samples. The resulting peptide fractions were desalted, and 100 fmol of a heavy isotope-labeled acK-peptide standard (m/z 626.8604⁺⁺; LVSSVSDLPacKR-¹³C₆¹⁵N₄) was added. acK peptides were immunoprecipitated using two anti-acK antibodies. Enriched peptides were separated and analyzed in duplicate by HPLC-MS/MS. Data were processed using Skyline to generate a MS/MS spectral library of the identified acK peptides. Precursor ion intensity chromatograms for one or more isotopes (M, M+1, M+2) of each peptide were integrated using Skyline MS1 Filtering and used for label-free quantitation across all samples after normalization to the peak area of the peptide standard.