Mitochondrial isolation and protein quantification

At sacrifice, livers were harvested and homogenized in a homogenization buffer containing 10 mM Trizma base-MOPS, 1 mM EGTA-Trizma base, and 200 mM sucrose supplemented with protease inhibitor. The liver homogenate was further processed to isolate the mitochondria after two steps of centrifugation, at 600×g for 10 min and 7000×g for 20 min sequentially. The resultant mitochondrial pellet was resuspended in mitochondrial lysis buffer. After six freeze/thaw cycles, the fraction was centrifuged at 18,000×g for 15 min to obtain the mitochondrial matrix. Protein estimation was performed using the bicinchoninic acid method (BCA protein assay kit; Thermo Scientific Pierce) according to the manufacturer’s recommendations. Semiquantitative analysis of PCCA and PCCB protein expression was achieved by capillary electrophoresis (Wes, ProteinSimple) based on the manufacturer’s recommendations. For detection of PCCA and PCCB, a polyclonal anti-PCCA antibody (Proteintech #21988-1-AP, 1:1000) and a polyclonal anti-PCCB antibody (Novus #NBP1-85886, 1:1000) were used, respectively. β-Actin, probed by a monoclonal anti-β-actin antibody (Sigma #A2228-200UL, 1:1000), served as a loading control for normalization. Absolute quantification of human PCCA and PCCB proteins was performed by LC-MS/MS. Mouse liver was homogenized in 100 mM ammonium bicarbonate and 8 M urea, and spiked with isotopically labeled signature peptides (natural C and N atoms on lysine were replaced by ¹³C and ¹⁵N isotopes; Thermo Scientific Pierce) for human WT PCCA (AQAVHPGYGFLSENK which does not recognize human PCCA A138T mutant) and PCCB (TVGIVGNQPK or DFFNYLPLSSQDPAPVR) as an internal standard. Protein was reduced, alkylated, and then digested with trypsin (Promega) at 37 °C for 15 h. After desalting, quantification was performed in a Thermo Easy 1200 nano-LC, QE plus Mass Spectrometer, as previously described¹. The lower limitations of quantification (LLOQs) were 26 and 16 ng/mg tissue for hPCCA and hPCCB proteins, respectively. Protein levels at pmol/mg were calculated from ng/mg based on the molecular weights of hPCCA and hPCCB proteins, which are 80,080 and 59,290 Daltons, respectively.