Sonic dismembranator

DMP cross-linked beads were gently well-mixed in 1 mL of bead wash buffer then pipetted in 100 μL aliquots (0.5 mg beads/sample) into Sarstedt 2.0 mL LB microtubes. Monkey heart tissue samples (20 mg to 200 mg) were weighed and transferred to a Sarstedt 2.0 mL LB microtube. Wash and dry the tube, accurately re-weigh it and record the amount of heart tissue. RIPA lysis buffer (500 μL) containing the protease inhibitor cocktail was added to heart tissue followed by approximately, 30–50 stainless steel beads (0.9–2.0 mm). Homogenization was conducted using the Bullet Blender Gold homogenizer (Next Advance, Troy, NY) at a speed of 10 for 5 min at 4 °C. Samples were then lysed completely by additional probe sonication on ice with 30 pulses at power 4 using a sonic dismembranator (Fisher Scientific, Pittsburgh, PA). The SILAC-hFXN-M internal standard solution (20 μL of 2 μg/mL, 40 ng) was then added to each sample and appropriate amounts of hFXN-M standards ng to 200 ng (4, 10, 15, 20, 30, 40, 80, 100, 150, 200 ng) in 5% BSA solution for preparation of the standard curve^{12 (link)}. Supernatants were removed from the DMP cross-linked beads and tissue and BSA standard samples added into the Sarstedt 2.0 mL LB microtubes containing the DMP cross-linked beads. Samples were then incubated with the beads at 4 °C overnight on the rotator.

All blood samples were thawed at room temperature, and 500 μL of each sample was mixed with 750 μL NP-40 lysis buffer (150 mM NaCl, 50 mM Tris/HCl pH 7.5, 0.5% Triton X-100, 0.5% NP-40, 1 mM DTT, 1 mM EDTA) containing protease inhibitor cocktail. The same amount of SILAC-labeled mature frataxin (20 ng) was spiked in each sample (calibrator, QC and whole blood) as an internal standard. Samples were lysed by probe sonication on ice for 30 pulses at power 5 using a sonic dismembranator (Fisher, Pittsburgh, PA, United States), followed by centrifugation at 17,000 g for 15 min at 4^°C. The supernatant was transferred from the pellet and incubated with pre-made DMP-crosslinked anti-frataxin protein G beads for immunoprecipitation.

Platelet and buffy coat from 1 mL blood and whole blood, erythrocytes and plasma from 100 μL were dissolved in 300 μL IP-lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 0.5% NP-40, 1 mM DTT) supplemented with 1x complete protease cocktail and incubated on ice for 15 min. 50 µg platelet lysates, 70 µg buffy coat lysate and whole blood, erythrocyte and plasma lysates each from 40 μL whole blood were spiked with a lysine and leucine stable isotope labeled frataxin internal standard (1 ng)^{22 (link)}. Samples were sonicated for 30 sec using a sonic dismembranator (Fisher) and centrifuged at 16,000 g using a bench top centrifuge for 15 min at 4 °C. The supernatant was applied to an Amicon Ultracel-50K filters (MilliporeSigma). The filters were spun at 4000 g, 4 °C for 20 min. The flow-through was transferred to Amicon Ultracel-3K filter units and spun at 4000 g, 4 °C for 40 min until the sample volume was concentrated to less than 50 μL. Samples were then subjected to western blot analysis and the results are shown in Fig. 1.

Metabolomic extraction protocol for fibroblasts was adapted from Guo et al. (22 (link)). A volume of 1 ml of cold 80% MeOH (−80°C), 40 μl of Metabolomics ISTD mix were added to each plate (see concentration for individual metabolites in supplemental Table S2). Cells were scraped and transferred to microcentrifuge tubes in ice. Samples were pulse-sonicated in ice with a sonic dismembranator (Fisher Scientific, Waltham, MA) for 30 s, incubated on ice for 10 min, and then pulse-sonicated again for 30 s. Samples were pelleted by centrifugation at 6,000 g for 5 min at room temperature. A volume of 500 μl of supernatant was moved to a clean microcentrifuge tube, dried down under nitrogen, and resuspended in 50 μl of 5% (w/v) 5-sulfosalicylic acid in water. Three microliter injections were used for LC-HRMS analysis with metabolomics method.