Composition, Structure and Function from Endopeptidease of Aplysia Egg Analyzed with Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry

Abstract

Endopeptidase of Aplysia egg (AEE) was purified on two column chromatography of both DEAE-52 cellulose and Sephadex G-150 for analysis of mass spectrometry. SDS-PAGE revealed the AEE purity and the molecular weight of its subunit at 39.0 kDa, called AEE 39. The results from matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) showed that AEE 39 consisted of single subunit type [M], indicating that the ratio of mass to change ( m/z) in AEE 39 was 12738.17, 18108.79 and 38221.42, providing molecular formula [M + 3H] 3+, [M + 2H] 2+ and [M + H] +. Using probe of insulin, a combined technology of metal cheater EDTA and MALDI-TOF-MS was employed to measure the molecular weight in AEE 39 and to identify its metal type, which showed an accurate molecular weight of 38221.42 Da and found metal zinc in AEE 39. AEE 39 has the capacity for degrading INS, which provided cleavage peptides measured with MALDI-TOF MS at m/z 1449.51, 2085.84, 4080.41 and 4165.42. Special software designed by the author was used to analyze the amino acids sequences of degradation produces and to identify the cleavage sites in INS. As a conclusion, it was found that the best cleavage site that degraded with AEE 39 was Leu-X (X: residues of amino acid) in INS, followed by Glu-X. In addition, the results indicated that Phen-X, Asn-X and Ser-X in INS can also be degraded by the endopeptidase. By comparing the molecular structures of both INS and attractin, it was found that one of the main functions in AEE 39 was responsible for degrading the attractin in eggs, which may play an important role in informational intercourse, recalling, recognizing, and mating. In addition, AEE 39 had another novel function for degrading Leu-Leu in acidic peptide. AEE 39 is a multifunctional ednopeptidase.