June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Determination of Fragments of SHLP2 in AMD Patients
Author Affiliations & Notes
  • Astghik Ghazaryan
    University of California Irvine, Irvine, California, United States
  • Mustafa Ozgul
    University of California Irvine, Irvine, California, United States
  • MARISABEL ANDRADE MELENDEZ
    University of California Irvine, Irvine, California, United States
  • Farid Jose Thomaz Neto
    University of California Irvine, Irvine, California, United States
  • Maria Cristina Kenney
    University of California Irvine, Irvine, California, United States
  • Baruch D Kuppermann
    University of California Irvine, Irvine, California, United States
  • Footnotes
    Commercial Relationships   Astghik Ghazaryan None; Mustafa Ozgul None; MARISABEL ANDRADE MELENDEZ None; Farid Thomaz Neto None; Maria Cristina Kenney Discovery Eye Foundation, Polly and Michael Smith, Iris and B. Gerald Cantor Foundation, NEI R01-EY027363, (MCK), NEI R21-EY033942-01 (MCK), Unrestricted Grant from Research to Prevent Blindness, Code C (Consultant/Contractor); Baruch Kuppermann Allegro Ophthalmics • Allergan • Genentech Inc • Ionis • IVERIC Bio • Novartis Pharmaceuticals • Regeneron Pharmaceuticals Inc • RegenXBio, Code C (Consultant/Contractor)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 2105. doi:
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      Astghik Ghazaryan, Mustafa Ozgul, MARISABEL ANDRADE MELENDEZ, Farid Jose Thomaz Neto, Maria Cristina Kenney, Baruch D Kuppermann; Determination of Fragments of SHLP2 in AMD Patients. Invest. Ophthalmol. Vis. Sci. 2023;64(8):2105.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Small humanin like peptide 2 (SHLP2) is a mitochondrial derived peptide that is coded from the mitochondrially encoded 16S rRNA (MT-RNR2) gene in mitochondrial DNA (mtDNA). SHLP2 contains 26 amino acids, with a sequence of MGVKFFTLSTRFFPSVQRAVPLWTN. Studies have shown that SHLP2 is associated with age related diseases and the serum SHLP2 levels decrease with aging. Previous studies in our laboratory showed that SHLP2 prevents loss of viable cells and mitochondria, increases the number of mtDNA copies, has anti-apoptotic functions, and reduces amyloid-β-induced cellular along with mitochondrial toxicity in age-related macular degeneration (AMD) cybrids.

The purpose of this study was to use ultra-performance liquid chromatography coupled to high resolution mass spectrometry (UPLC-HRMS) to analyze the human plasma samples and identify numerous SHLP2 fragments and their oxidized forms.

Methods : Five plasma samples from AMD patients were frozen at -80oC until further use. After thawing and diluting the plasma samples to 20% acetonitrile, each sample was filtered using Amicon Ultra-4 10kDa ultrafiltration devices for 30 minutes, at 4oC, at 17000g. Using LC-MS grade water, the filtered samples were diluted 10 times. Immediately, the solutions were analyzed by UPLC-HRMS to determine the degradation products of the SHLP2 peptide in each plasma samples. Collected data from UPLC-HRMS instrument were analyzed to identify the amino acid sequences of SHLP2 fragments in the AMD plasma samples using BiopharmaLynxTM software.

Results : Our study showed that human plasma samples contain degraded endogenous SHLP2 peptides. In human plasma samples from AMD patients, we have found 26 different SHLP2 fragments and 2 oxidized SHLP2 fragments as well as their intensity levels of each plasma sample.

Conclusions : With advanced UPLC-HRMS technologies, in plasma samples from AMD patients, fragments of SHLP2 have been found. In addition, identified amino acid sequences of SHLP2 fragments and their intensity levels were determined using BiopharmaLynxTMsoftware. These results can guide researchers to plan better experimentalapproaches to discover possible critical biological functions of SHLP2 fragments

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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