Abstract
Purpose :
MOTS-c peptide is one of the mitochondrial derived peptides which represent a new class of biologically active molecules with potential to protect retinal cells from oxidative stress associated with retinal pathology. State-of-the-art techniques can be used to analyze stability of intact MOTS-c peptide and identify MOTS-c fragments for possible use in future therapeutic investigation of age-related macular degeneration.
Methods :
Stability analyses was done by High-Resolution Mass Spectrometry (HRMS). During this process the MOTS-c solutions with a concentration of 12.5 μg/ml were stored at 4°C and 37°C for 30 days.
Mass spectrometric analysis was performed using Xevo G2-XS Quadrupole Time-of-Flight mass spectrometer (HRMS) coupled to UPLC. The UPLC method used 30 minutes linear gradient at 0.3 mL/min from 97% A to 97% B where A is 0.1% Formic Acid in water and B is 100% Acetonitrile. For HRMS analysis, positive electrospray ionization mode was utilized. A capillary transfer temperature of 300°C and a spray voltage of 3.0 kV were used to accomplish ionization. A resolution of 30,000 Full Width at Half Maximum was used for a full scan experiment within a range of 100–2000 m/z as well as 15,000 FWHM with an isolation window adjusted to 2.0 m/z for. Leucine Enkephalin was used as a lock mass for nominal mass correction, and a CsNaI ladder was used to calibrate the detector.
Results :
In MOTS-c peptide with molecular weight of 2173.11g/mol, the main ions of +5, +4 and +3 were formed at 435.84, 544.54 and 725.72 m/z (Figure 1). In MOTS-c, oxidation of methionine remained constant upon incubation of MOTS-C in water for 30 days at 37oC. Intact MOTS-c peptides without oxidation were detected at the highest intensities. HRMS spectrums of MOTS-c stability in HPLC water are presented in Figures.
Conclusions :
It is the first time that 1-month stability properties of MOTS-c peptide and its oxidation and degradation products have been analyzed in detail using advanced HRMS technologies. The data suggest that intact MOTS-c peptides in HPLC water represent a more stable form than Humanin-G peptides in water. The results may help researchers design better in vitro and in vivo experimental parameters to further understand the critical role of MOTS-c in physiological conditions and human diseases.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.