DOI: 10.5061/dryad.n02v6wx2p
title: Mass spectrometry data for: A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics

description: Background: Mitochondria have a central role in cellular functions, aging
 and in certain diseases. They possess their own genome, a vestige of their
 bacterial ancestor. Over the course of evolution, most of the genes of the
 ancestor have been lost or transferred to the nucleus. In humans, the
 mtDNA is a very small circular molecule with a functional repertoire
 limited to only 37 genes. Its extremely compact nature with genes arranged
 one after the other and separated by short non-coding regions suggests
 that there is little room for evolutionary novelties. This is radically
 different from bacterial genomes, which are also circular but much larger,
 and in which we can find genes inside other genes. These sequences,
 different from the reference coding sequences, are called alternative open
 reading frames or altORFs, and they are involved in key biological
 functions. However, whether altORFs exist in mitochondrial protein-coding
 genes or elsewhere in the human mitogenome has not been fully addressed.
 Results: We found a downstream alternative ATG initiation codon in the +3
 reading frame of the human mitochondrial nd4 gene. This newly
 characterized altORF encodes a 99-amino acids long polypeptide, MTALTND4,
 which is conserved in primates. Our custom antibody, but not the
 pre-immune serum, was able to immunoprecipitate MTALTND4 from HeLa cell
 lysates, confirming the existence of an endogenous MTALTND4 peptide. The
 protein is localized in mitochondria and cytoplasm and is also found in
 the plasma, and it impacts cell and mitochondrial physiology. Conclusions:
 Many human-mitochondrial-translated ORFs might have so far gone unnoticed.
 By ignoring mtaltORFs, we have underestimated the coding potential of the
 mitogenome. Alternative mitochondrial peptides such as MTALTND4 may offer
 a new framework for the investigation of mitochondrial functions and
 diseases.
description: HeLa cells (n=3 replicates)
 were washed with PBS and harvested in a lysing buffer containing 10 mM
 HEPES, 150 mM NaCl and a cocktail of protease inhibitors (benzamidine,
 PMSF, aprotinin and leupeptin). Cells were then lysed with a sonic
 dismembrator sonicator (Fisher) 3 times for 30 seconds before adding 100
 µl of Triton X-100 10%. Cell lysates were incubated on ice for 20 minutes
 and then centrifuged for 15 minutes at 15000 RPM at 4˚C. The supernatant
 was kept. 20 µl of protein A agarose was added to 1 ml of samples and
 incubated at 4˚C for 1 hour. Samples were then centrifuged at 15000 rpm
 for 1 minute at 4˚C and the supernatant was kept. 20 µl of rabbit
 anti-MTALTND4 antibody and 20 µl of protein A agarose were added to the
 samples before incubating at 4˚C overnight. 20 µl of rabbit pre-immune
 serum was used as control. Samples were washed 6 times with lysing buffer.
 Samples were then sent to the proteomics platform of the McGill University
 Health Center (MUHC) Research Institute for mass spectrometry analysis.
 Samples were loaded onto a
 single stacking gel band to remove lipids, detergents and salts. The
 single gel band containing all proteins was reduced with DTT, alkylated
 with iodoacetic acid and digested with trypsin. 2 µg of extracted peptides
 were re-solubilized in 0.1% aqueous formic acid and loaded onto a Thermo
 Acclaim Pepmap (Thermo, 75µM ID X 2 cm C18 3 µM beads) precolumn and then
 onto an Acclaim Pepmap Easyspray (Thermo, 75 µM X 15 cm with 2 µM C18
 beads) analytical column separation using a Dionex Ultimate 3000 uHPLC at
 250 nl·min<sup>-1</sup> with a gradient of 2–35% organic (0.1%
 formic acid in acetonitrile) over 3 hours. Peptides were analyzed using a
 Thermo Orbitrap Fusion mass spectrometer operating at 120,000 resolutions
 (FWHM in MS1) with HCD sequencing (15,000 resolution) at top speed for all
 peptides with a charge of 2+ or greater. The raw data were converted into
 *.mgf format (Mascot generic format) for searching using the Mascot 2.6.2
 search engine (Matrix Science) against Human Uniprot sequences (2022). The
 database search results were loaded onto Scaffold Q+ Scaffold_4.9.0
 (Proteome Sciences) for statistical treatment and data
 visualization (Table
 S4).