Analysis of HNF14 Peptide using Basic Local Alignment Search Tool and High-Resolution Mass Spectrometry

Abstract

Purpose : The HNF14 sequence (LLLTSEIDLPVKRR) derived from the degradation product of humanin, a mitochondrial-derived peptide. Our research primarily focused on two key aspects of the Humanin Fragment 14 peptide (HNF14).

First, we used various tools such as basic local alignment search tool (BLAST), multiple alignment sequence viewer, and blast tree view to assess sequence similarities across various organisms. Second, we investigated the stability of the HNF14 peptide in LC-MS grade water and PBS in long-term periods (8 months) at 37^oC.

Our objective is to shed light on the potential therapeutic properties of the HNF14, particularly in mitochondrial biology and its associated diseases. Our research contributes to the growing field centered on mitochondrial-derived peptides and their therapeutic applications.

Methods : The HNF14 sequence was subjected to a BLAST search using the NCBI BLAST server. The phylogenetic tree was visualized using the Blast Tree View Widget available on the NCBI BLAST website.

For long-term stability analyses, 20 μM HNF14 in LC-MS grade water and phosphate-buffered saline (PBS) were maintained at 37°C for 8 months. The degradation and oxidation patterns of the stored HNF14 solutions were evaluated using Ultra-Performance Liquid Chromatography – High Resolution Mass Spectrometry (Waters Xevo G2-Xs Qtof).

Results : Upon conducting a BLAST search using HNF14 sequence, multiple hits were obtained (Table 1). The sequence exhibited high similarity from various organisms, suggesting a potential functions of this peptide across different species.

For long-term stability analyses, we found that HNF14 in PBS degrades more slowly than HNF14 in LC-MS grade water at 37 °C. The highest intensities of HNF14 were found in PBS solution at 37 °C for 11 months. However, multiple different HNF14 were identified in LC-MS grade water at 37 °C for 11 months, suggesting that HNF14 is more unstable in LC-MS grade water.

Conclusions : High similarity of the HNF14 sequence was found in various organisms, suggesting the potential importance in cellular survival and functionality. These observations suggest that the peptide, even as a fragment of humanin peptide, might still exhibit some of the characteristic functions of humanin peptide or even exhibit unique bioactivities of its own.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.