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. 2023 Mar 11;13(3):515.
doi: 10.3390/biom13030515.

Stability Determination of Intact Humanin-G with Characterizations of Oxidation and Dimerization Patterns

Mustafa Ozgul  1 , Anthony B Nesburn  1   2 , Nader Nasralla  3 , Benjamin Katz  4 , Enes Taylan  5 , Baruch D Kuppermann  1 , Maria Cristina Kenney  1   6
Affiliations

Stability Determination of Intact Humanin-G with Characterizations of Oxidation and Dimerization Patterns

Mustafa Ozgul et al. Biomolecules. .

Abstract

Humanin is the first identified mitochondrial-derived peptide. Humanin-G (HNG) is a variant of Humanin that has significantly higher cytoprotective properties. Here, we describe the stability features of HNG in different conditions and characterize HNG degradation, oxidation, and dimerization patterns over short-term and long-term periods. HNG solutions were prepared in high-performance liquid chromatography (HPLC) water or MO formulation and stored at either 4 °C or 37 °C. Stored HNG samples were analyzed using HPLC and high-resolution mass spectrometry (HRMS). Using HPLC, full-length HNG peptides in HPLC water decreased significantly with time and higher temperature, while HNG in MO formulation remained stable up to 95% at 4 °C on day 28. HNG peptides in HPLC water, phosphate-buffered saline (PBS) and MO formulation were incubated at 37 °C and analyzed at day 1, day 7 and day 14 using HRMS. Concentrations of full-length HNG peptide in HPLC water and PBS declined over time with a corresponding appearance of new peaks that increased over time. These new peaks were identified to be singly oxidized HNG, doubly oxidized HNG, homodimerized HNG, singly oxidized homodimerized HNG, and doubly oxidized homodimerized HNG. Our results may help researchers improve the experimental design to further understand the critical role of HNG in human diseases.

Keywords: degradation products; high-performance liquid chromatography (hplc); high-resolution mass spectrometry (hrms); oxidations; peptides; stability.

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Conflict of interest statement

M.C.K.: Her research on mitochondria has been supported by Discovery Eye Foundation (DEF). She serves as a member of the Board for DEF. The University of California, Irvine has reviewed and approved the terms of this arrangement in accordance with conflicts-of-interest policies. M.C.K.: Collaborations with Allegro, Ophthalmics, LLC and Almon Therapeutics, Inc. B.D.K: CLINICAL RESEARCH: Alcon, Alimera, Allegro, Allergan, Apellis, Clearside, Genentech, GSK, Ionis, jCyte, Novartis, Regeneron, ThromboGenics; CONSULTANT: Alimera, Allegro, Allergan, Cell Care, Dose, Eyedaptic, Galimedix, Genentech, Glaukos, Interface Biologics, jCyte, Novartis, Ophthotech, Regeneron, Revana, Theravance Biopharma.

Figures

Figure 5
Figure 5
(A) Amino acid sequence and m/z ratio of DM-HNG are represented. (B) Representative HRMS precursor ion mass spectra of DM-HNG in PBS. (C) Amino acid sequence and m/z ratio of SOX-DM-HNG are represented. (D) Representative HRMS precursor ion mass spectra of SOX-DM-HNG in PBS. (E) Amino acid sequence and m/z ratio of DOX-DM-HNG are represented. (F) Representative HRMS precursor ion mass spectra of DOX-DM-HNG in PBS.
Figure 6
Figure 6
Ion chromatogram results of incubated HNG at 37 °C for 1 day in PBS (A), HPLC-grade water (B), and MO formulation (C).
Figure 7
Figure 7
Ion chromatogram results of incubated HNG at 37 °C for 7 days in PBS (A), HPLC-grade water (B), and MO formulation (C).
Figure 8
Figure 8
Ion chromatogram results of incubated HNG at 37 °C for 14 days in PBS (A), HPLC-grade water (B), and MO formulation (C).
Figure 9
Figure 9
Overlay ion chromatogram (19 min–22.5 min) results of incubated HNG at 37 °C in PBS, HPLC-grade water and MO formulation at day 1 (A), at day 7 (B), and at day 14 (C).
Figure 10
Figure 10
Overlay ion chromatogram (22.5 min–24 min) results of incubated HNG at 37 °C in PBS, HPLC-grade water and MO formulation at day 1 (A), at day 7 (B), at day 14 (C).
Figure 11
Figure 11
Evaluation of intensity changes of SOx-HNG (A), DOX-HNG (B) at day 1, 7 and 14.
Figure 12
Figure 12
Evaluation of intensity changes of homodimerized form of HNG (A), SOX-DM-HNG (B), DOX-DM-HNG (C) at day 1, 7 and 14.
Figure 13
Figure 13
Measurements of HNG in HPLC-grade water (Upper Panel A) and MO formulation (Lower Panel B) at 4 °C for 11 months using HRMS. UPPER PANEL A, (A) Amino acid sequence and m/z ratio of dimerized form HNG is represented; (B,C) Representative ion chromatogram and HRMS product ion mass spectra of HNG in HPLC water, respectively. LOWER PANEL B, (D) Amino acid sequence and m/z ratio of HNG is represented; (E,F) Representative ion chromatogram and HRMS product ion mass spectra of HNG in MO formulation.
Figure 1
Figure 1
(A) The NMR structure of HNG peptide (PDB identifier 2GD3 Chain 8) is visualized using the UCSF Chimera program. The length and width of HNG are ~4 nm and ~1.5 nm, respectively. The sequence of full-length HNG is shown. (B) The diagram shows the net charge of HNG as a function of pH. (C) Physiochemical properties of HNG.
Figure 2
Figure 2
Ion chromatogram results of HNG in HPLC-grade water (Left Panel) and MO formulation (Right Panel). LEFT PANEL: HNG in Water. (A) Day 0 at RT; (B) 3rd day +37 °C; (C) 3rd day +4 °C; (D) 7th day +37 °C; (E) 7th day +4 °C. (F) 14th day +37 °C; (G) 14th day +4 °C; (H) 28th day +37 °C; (I) 28th day +4 °C. RIGHT PANEL: HNG in MO formulation. (J) Day 0 at RT; (K) 3rd day +37 °C; (L) 3rd day +4 °C; (M) 7th day +37 °C; (N) 7th day +4 °C; (O) 14th day +37 °C; (P) 14th day +4 °C; (R) 28th day +37 °C; (Q) 28th day +4 °C. RT, room temperature: HNG Humanin-G; Pd: Product.
Figure 3
Figure 3
Stable HNG peptide concentration changes over time in HPLC water versus MO formulation at 4 °C and 37 °C.
Figure 4
Figure 4
(A) Amino acid sequence and m/z ratio of the full-length HNG are represented. (B) Representative HRMS precursor ion mass spectra of HNG in PBS. (C) Amino acid sequence and m/z ratio of SOX-HNG are represented. (D) Representative HRMS precursor ion mass spectra of SOX-HNG in PBS. (E) Amino acid sequence and m/z ratio of DOX-HNG HNG are represented. (F) Representative HRMS precursor ion mass spectra of DOX-HNG in PBS.
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