Proteomic and scanning electron microscopic analysis of submandibular sialoliths

Abstract

Several theories have been proposed regarding the genesis of sialoliths, including the organic core theory, which suggests epithelial or bacterial etiology originating in the central core. Our aim was to use novel methodologies to analyze central areas (the core) of calculi from sialolithiasis patients. The structures of the halves of six submandibular salivary stones were analyzed by scanning electron microscopy (SEM). After structural analysis, from the other six halves, samples from the central parts of the core and peripheral parts of the core were digested with trypsin and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The peptide mass fingerprints were compared with the results of in silico digestion. SEM analysis of the sialoliths showed that organic structures (collagen/fibrous-like structures, bacterial fragments) were visible only outside of the core in the concentric layers of external areas, but not in the core area. The mass spectrometry (MS)/MS post-source decay experiments were completed from the four, most intense signals observed in the MS spectrum and human defensin was proven to be present in three of the examined samples, originated from the peripheral region of three cores. Although proteomic analysis demonstrated defensin protein in the peripheral region of the core in three sialoliths, SEM failed to prove organic structures in the core. New investigation modalities still cannot prove organic structures in the core, henceforward challenging the organic core theory.