Development of a mass spectrometry based detection method for the mitochondrion‐derived peptide MOTS‐c in plasma samples for doping control purposes

Abstract

Rationale The mitochondrial open reading frame of 12S rRNA type‐c (MOTS‐c) peptide was recently discovered and described to control metabolic homeostasis through AMPK activation along with AICAR accumulation. Consequently, it appears advisable to monitor the potential use of synthetic MOTS‐c in sports, and a detection method suitable for sports drug testing purposes is necessary. Methods For the detection of MOTS‐c in doping control plasma samples, a test method employing liquid chromatography and mass spectrometry (LC/MS) was developed. Following optimization, the assay was comprehensively validated and additional parameters such as the (long‐term) stability and in vitro metabolism of the peptide were evaluated. In order to determine endogenous MOTS‐c reference limits, the results generated by LC/MS‐based detection were compared with those obtained with a commercially available enzyme‐linked immunosorbent assay (ELISA). Results The LC/MS‐based test method was fully validated for quantitative results interpretation according to the World Anti‐Doping Agency's International Standard for Laboratories (WADA's ISL). It was found to be specific and sensitive, enabling a lower limit of detection (LLOD) for hMOTS‐c in plasma at 100 pg/mL. Following optimization, animal MOTS‐c analogues and four metabolites as well as two oxidation products were implemented. However, endogenous levels of a reference population of 20 healthy subjects studied by ELISA experiments (45.9–218.5 ng/mL) could not be confirmed by LC/MS. Conclusions A mass spectrometric detection assay for MOTS‐c in human plasma samples was developed and successfully validated according to WADA's ISL, providing an additional tool for future doping control purposes. Besides MOTS‐c, the assay also includes four in vitro derived metabolites and two oxidation products, which might further improve the traceability of the drug. The analytical approach was compared with a commercially available ELISA, and considerable differences in measured MOTS‐c levels were observed.

MOTS-c is an exercise-induced mitochondrial-encoded regulator of age-dependent physical decline and muscle homeostasis

“…In fact, we previously reported that systemic MOTS-c treatment reversed diet-induced obesity and diet-and age-dependent insulin resistance in mice 15 . We tested if MOTS-c functions as a mitochondrial-encoded regulator of physical capacity and performance 2,22,23 in young (2 mo. ), middle-aged (12 mo.…”

Extension of Mitogenome Enrichment Based on Single Long-Range PCR: mtDNAs and Putative Mitochondrial-Derived Peptides of Five Rodent Hibernators

“…A unique feature of MDPs represents the combination of intra-mitochondrial transcription with cytoplasmic translation according to the nuclear genetic code ( Cobb et al, 2016 ; Lee et al, 2016 ). In case of circulating cell-free MOTS-c, mass spectrophotometry delivered gold-standard proof for MDP expression ( Knoop et al, 2019 ; Reynolds et al, 2021 ). MDPs regulate apoptosis and metabolic homeostasis, insulin sensitivity and obesity, reduce oxidative stress and inflammation, extend life expectancy, signal retrograde communication of mitochondria with the nucleus, protect against cancer and Alzheimer´s disease, show decreasing expression with age ( Cobb et al, 2016 ; Kim et al, 2017 ; Lu et al, 2019 ) and bear considerable potential to be misused as a performance-enhancing drug in elite sports ( Thevis and Schänzer, 2016 ).…”

Annual banned‐substance review – Analytical approaches in human sports drug testing

“…Other drug candidates, however, might influence the endogenous production and/or accumulation of AICAR such as the “mitochondrial open reading frame of 12S rRNA type‐c” (MOTS‐c) peptide, which consequently necessitate consideration in proactive antidoping research. Knoop et al focused on targeting MOTS‐c and in vitro ‐generated metabolic products in human plasma and serum by means of LC–MS/MS . A volume of 100 μL of plasma (or serum) was enriched with stable isotope‐labeled MOTS‐c as internal standard, and plasma proteins were precipitated by the addition of acetonitrile.…”