Tissue culture, acquisition, and analysis of pulse-SILAC mass spectrometry data

Pulse-SILAC experiment was performed as previously described (Hock et al., 2020 ) with modifications. Cells were cultured in triplicate in DMEM high glucose) supplemented with 10% (vol/vol) fetal calf serum, penicillin–streptomycin, 50 μg/ml uridine, and 1 μg/ml tetracycline at 37 °C under an atmosphere of 5% CO₂. Chloramphenicol (CAP; Sigma) was added at 50 µg/ ml for 24 h before pulse to inhibit mitochondrial translation. The next day, media was replaced with DMEM for SILAC (Thermo Fisher Scientific) supplemented with 10% (vol/vol) dialyzed fetal calf serum (dFCS; Thermo Fisher Scientific), penicillin/streptomycin (Life Technologies), 1 mM sodium pyruvate (Life Technologies), 1X Glutamax (Life Technologies), 3.5 g/l glucose, 50 µg/ml uridine, 1 µg/ml tetracycline, 600 mg/ml l-proline (Merck), 146 mg/ml -¹³C₆¹⁵N₂-l-lysine-HCl, and 42 mg/l -¹³C₆¹⁵N₂-l-arginine-HCl (Silantes). Time points were collected at 1, 3, and 4 h post–heavy SILAC media incubation. Cells were washed twice with PBS, pelleted at 500 × g, and frozen at −80°C until use.

Mitochondrial isolation was performed as previously described (Acín-Pérez et al., 2008 ) with modifications. Briefly, cell pellets were resuspended in buffer A (83 mM sucrose, 10 mM HEPES, pH 7.2) and homogenized with 10 strokes using a KIMBLE glass Dounce homogenizer (Sigma). An equal amount of buffer B (250 mM sucrose, 30 mM HEPES, pH 7.2) was added and centrifuged at 1000 × g for 5 min to remove cell debris and unbroken cells. Mitochondria were collected from the supernatant by centrifuging at 10,000 × g for 2 min. Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific) and normalized to 50 μg for quantitative proteomics preparation. Mitochondrial pellets were prepared for mass spectrometry using the same method as described under Quantitative mass spectrometry and data analysis.

Peptides were analyzed on an Orbitrap Eclipse Tribid mass spectrometer (Thermo Fisher Scientific). LC coupled MS/MS was carried out with a nanoESI interface in conjunction with an Ultimate 3000 RSLC nanoHPLC (Dionex Ultimate 3000). The LC system was equipped with an Acclaim Pepmap nano-trap column (Dionex-C18; 100 Å, 75 μ × 2 cm) and an Acclaim Pepmap RSLC analytical column (Dionex-C18; 100 Å, 75 μM × 50 cm). The tryptic peptides were injected to the trap column at an isocratic flow of 5 μl/min of 2% ACN containing 0.1% (vol/vol) formic acid for 5 min applied before the trap column was switched in-line with the analytical column. The eluents were 5% DMSO in 0.1% vol/vol formic acid (solvent A) and 5% DMSO in 100% vol/vol ACN and 0.1% vol/vol formic acid (solvent B). The flow gradient was 1) 0–6 min at 3% B, 2) 6–95 min, 3–23% B, 3) 95–105 min, 23–40% B, 4) 105–110 min, 40–80% B, 5) 110–115 min, 80–80% B, 6) 115–117 min, 80–3% B and equilibrated at 3% B for 10 min before the next sample injection.

For this experiment, the Orbitrap Eclipse mass spectrometer was operated in the data-dependent mode with a targeted inclusion list containing predicted peptides from the 13 mitochondrial DNA-encoded proteins. The inclusion list consists of mass/charge (m/z) and charge (z) or tryptic peptides (endogenous and SILAC labeled) predicted from in silico digest of target proteins using the Skyline software (MacLean et al., 2010 ). In addition, the inclusion list also contained peptides that have been previously observed in public data depositories through the PeptideAtlas site (Deutsch et al., 2008 ), reference peptides from ProteomicsDB (Schmidt et al., 2018 ), and the present study.

The acquisition method was created with the Orbitrap Tribid Tune version 3.3 acquisition software. The full MS1 spectra from 375 to 1500 m/z was acquired in positive mode at 120,000 resolution. Two scan priorities were created post–MS1 scans. The first priority was given to precursors that fulfill the mass and charge criteria. Precursors were then isolated using isolation window of 1.6 m/z and precursors fragmented using fixed normalized collision energy of 30. MS2 resolution was at 15,000, AGC target at 5e⁵, and maximum IT time of 100 ms. The second scan priority is activated when no matching mass from the target list is identified. Precursors were isolated with the same parameters with the difference of maximum IT of 22 ms. Dynamic exclusion was set to be 30 s.

Raw files were processed using the MaxQuant platform (version 1.6.10.43) (Cox and Mann, 2008 ) and searched against the UniProt human database (42,434 entries, June 2019) using default settings for a SILAC experiment with “label min. ratio count” set to 1 and match between runs enabled. From the proteinGroups.txt output file, mtDNA-encoded proteins were filtered by gene name and identified by at least two peptides. Heavy intensities were log₂ transformed in Prism (version 8.4.3; GraphPad) and normalized to the maximum value detected in the WT line at 4 h pulse. The means from three experiments were plotted over time using Prism along with the SD. Statistical significance using t test was performed in Prism using the two-stage step-up method of Benjamini et al. (2006) and false discovery rate (FDR) of 1%.