What Is MOTS-c? The 16-Residue Mitochondrial Peptide

MOTS-c is a peptide of sixteen amino acid residues, Met-Arg-Trp-Gln-Glu-Met-Gly-Tyr-Ile-Phe-Tyr-Pro-Arg-Lys-Leu-Arg (MRWQEMGYIFYPRKLR), whose sequence is encoded by a short open reading frame inside the mitochondrial gene for 12S ribosomal RNA. As a research material it is a synthetic linear peptide with the molecular formula C101H152N28O22S2, an average molecular weight of 2174.6 g/mol and the CAS number 1627580-64-6.

This page covers MOTS-c as a molecule: where its sequence comes from, what the sequence implies for its formula, mass and charge, which chemical features matter in analysis, and what to check on a certificate of analysis.

A peptide encoded in a ribosomal RNA gene

The mitochondrial genome encodes a small set of proteins, the transfer RNAs and the two ribosomal RNAs of the mitochondrial ribosome. The gene for the smaller of those ribosomal RNAs, the 12S rRNA, carries the symbol MT-RNR1 ("mitochondrially encoded 12S rRNA") in the HGNC gene nomenclature, which lists MOTS-c among the gene's alias symbols. The gene's product is an RNA rather than a protein, but within its sequence lies a short open reading frame (ORF), and the name MOTS-c refers to that origin: a mitochondrial open reading frame of the 12S rRNA.

In the human mitochondrial reference sequence (RefSeq NC_012920.1), the 12S rRNA gene spans positions 648 to 1601 and the reading frame occupies positions 1343 to 1393. It is 51 nucleotides long: sixteen codons followed by a TAG stop codon. Substance registries such as PubChem list the compound as a mitochondria-derived peptide.

Two genetic codes, one sequence

Vertebrate mitochondria use a genetic code that differs from the standard one in a few codons, and two of those differences fall inside this reading frame. In the standard code, AGG and AGA both encode arginine. In the vertebrate mitochondrial code, as tabulated by NCBI (translation table 2), they are assigned as stop codons. The MOTS-c reading frame uses AGG at codon 2 and AGA at codon 13, so the published sixteen-residue sequence is the standard-code reading of the frame. Read with table 2, the frame would end after its first codon. The third arginine, at position 16, is encoded by CGA, which means arginine in both codes.

Written in full with its unmodified termini, the peptide is H-Met-Arg-Trp-Gln-Glu-Met-Gly-Tyr-Ile-Phe-Tyr-Pro-Arg-Lys-Leu-Arg-OH. The conventions behind this notation are explained in peptide sequence notation.

Composition, formula and mass

The sixteen residues are three arginines, two methionines and two tyrosines, plus one each of tryptophan, glutamine, glutamic acid, glycine, isoleucine, phenylalanine, proline, lysine and leucine. Joining sixteen amino acids through fifteen peptide bonds removes fifteen molecules of water, which gives the formula C101H152N28O22S2. Both sulfur atoms come from the two methionine side chains.

Two properties follow directly from the composition:

• A basic peptide. Three arginines, one lysine and the N-terminal amine can each carry a positive charge, against one glutamic acid and the C-terminal carboxyl. At neutral pH the net charge is about +3.
• A UV chromophore at 280 nm. One tryptophan and two tyrosines absorb near 280 nm. Using the standard residue values of 5,500 M⁻¹ cm⁻¹ for tryptophan and 1,490 M⁻¹ cm⁻¹ for tyrosine (Pace et al., 1995) gives a calculated molar absorption coefficient of 8,480 M⁻¹ cm⁻¹, a useful figure for UV-based measurements of the peptide.

Chemical features to watch in analysis

A few residues in MOTS-c are known sites of chemical change, and a good analysis checks for them.

• Methionine oxidation. Methionine at positions 1 and 6 can oxidise to methionine sulfoxide, adding 15.995 Da each. Oxidised forms appear at +16 and +32 Da; in the doubly charged ion series they sit about 8 and 16 m/z units above the parent peak. Sulfoxides are more polar than methionine, so these forms usually elute slightly earlier in reversed-phase HPLC.
• Tryptophan oxidation. The indole ring of tryptophan at position 3 can also oxidise, giving further +16 and +32 Da species that mass spectrometry alone cannot always tell apart from methionine oxidation. MS/MS fragments locate the modified residue.
• Glutamine at position 4. Its side-chain amide can deamidate to glutamic acid, a change of only +0.984 Da that is easy to miss without high-resolution mass spectrometry. Because glutamine is not at the N-terminus, pyroglutamate formation does not apply.
• Counter-ions. With up to five positively charged groups, MOTS-c is normally isolated as a salt, commonly with trifluoroacetate from purification; PubChem lists a trifluoroacetate salt among its synonyms. Part of the powder's mass is therefore counter-ion and water, so its net peptide content is lower than its gross weight.

How MOTS-c is analysed

Reading the electrospray spectrum is simple arithmetic. Each peak in the charge-state series has m/z = (M + z × 1.00728) / z, where M is the neutral mass, z the charge and 1.00728 the mass of a proton. Taking the triply charged ion at m/z 725.377: 3 × 725.377 − 3 × 1.00728 = 2173.109 Da, which matches the calculated monoisotopic mass within rounding. The 2+ and 4+ peaks should give the same answer, and a peak that does not fit the series points to a different species.

Research material of this kind is made by chemical synthesis, and a sixteen-residue chain is well within the routine range of solid-phase peptide synthesis. The by-products that method can leave are sequences missing one residue or cut short. With three arginines in the chain, a sequence missing one arginine, 156.101 Da below the parent mass, is one of the species to check for in a MOTS-c spectrum.

More detail on each method is in the guides to HPLC peptide purity and mass spectrometry for peptide identity.

Certificates, specification and storage

For our MOTS-c, the purity specification is ≥99% (HPLC, supplier specification), and certificate status is shown on every product page. When reading any certificate for MOTS-c, check that the sequence and the observed mass match the values above, that the ion type or deconvolution is stated, that the HPLC method gives its detection wavelength, and that the batch number and analysis date match the vial.

MOTS-c is supplied as a lyophilised solid in a sealed vial. Store the sealed vial at 2–8 °C for short-term storage, or at −20 °C and below for long-term storage, protect it from light and avoid repeated freeze-thaw cycles. With two methionines and a tryptophan, keeping air, light and moisture away from the solid matters more than for peptides without these residues.

MOTS-c belongs to the Mitochondrial Compounds research area. It is catalogued there with two compounds of different chemical classes: SS-31, an aromatic-cationic tetrapeptide, and NAD+, a dinucleotide coenzyme. Terms such as monoisotopic mass and open reading frame are defined in the glossary.