Abstract
Purpose :
State-of-the-art techniques can be used to identify Humanin-G (HNG) fragments and dimers for possible use in future therapeutic investigation in age-related disease such as age-related macular degeneration, Alzheimer’s disease and cancer.
Methods :
For long-term stability analyses, HNG solutions were stored for 11 months at 4°C. For High-Resolution Mass Spectrometry (HRMS) studies, samples with a concentration of 30 μM HNG in HPLC water were used to analyze 11-month-old HNG products.
Mass spectrometric analysis was performed using Xevo G2-XS Quadrupole Time-of-Flight (HRMS) mass spectrometer coupled to UPLC. The UPLC method was run on a Water BEH C4 column using a 25 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 (FWHM) was used for a full scan experiment within a range of m/z 100–2000 in addition to 15,000 FWHM with an isolation window adjusted to m/z 2.0 for. Leucine Enkephalin was used as a lock mass for nominal mass correction, and a CsNaI ladder was used for detector calibration.
Results :
The HRMS spectrum of Humanin-G degraded molecules and humanin-G homodimers are presented in Figure 1A and Table Peptide Sequences (PS). In Humanin-G, disulfide dimerization was observed upon incubation of Humanin-G in HPLC water after 11 months at +4oC constant. Humanin-G homodimers (molecular mass 5312), the main ions were +9, +8, +7, +6, +5, charged states at m/z 532.31, 591.23, 665.01, 759.85, 886.34, 1063.40, (Fig. 2, Table PS). Humanin-G homodimers were detected at the highest intensities. HRMS detected other dimerized Humanin-G fragments (Fig. 1, 2 and Table PS). Dimerized Humanin-G fragments are shown in Figure 3 and Table PS.
Conclusions :
For the first time, long-term stability properties of HNG peptide and its degradation products have been analyzed in detail using advanced HRMS technologies. Our data suggest that Humanin-G homodimers represent a more stable form than a single Humanin-G peptide. Our results may help researchers design better in vitro and in vivo experimental parameters to further understand the critical role of HNG in physiological conditions and human diseases.
This is a 2021 ARVO Annual Meeting abstract.