Evaluation of method parameters for sound undecalcified dental enamel proteomics using liquid chromatography-mass spectrometry

Objective: This study aims to validate a methodology for analyzing undecalcified, sound dental enamel proteomics using Liquid Chromatography-Mass Spectrometry (LC-MS). The study evaluates various parameters, including the impact of dental root coverage on protein contamination, the efficacy of protease inhibitors during enamel sample preparation, repeatability of LC-MS measurements on dental enamel, and statistical analysis. The study also assesses the effectiveness of combined trypsin and semi-trypsin searches in Mascot for obtaining additional protein identification data. Design: Sound dental enamel was removed using a wet grinding technique, then digested with trypsin and labeled with TMT prior to LC-MS analysis. The resulting proteomes were matched against the Homo sapiens Swissprot Database, with searches in Mascot performed using both trypsin and semitrypsin. Statistical methods were employed to analyze the data. Results: The study found that covering dental roots with composite during dental enamel microdissection is advisable, while using protease inhibition during microdissection may not be fully supported. The proteomic analyses demonstrated statistical repeatability and reliability, with consistent and reproducible proteomic data obtained from healthy dental enamel. Furthermore, employing both trypsin and semitrypsin searches in Mascot provided additional proteomic information. Conclusions: Overall, this study validates a methodology for analyzing undecalcified, sound dental enamel proteomics using LC-MS, and provides insights into various factors that can affect the quality and reliability of proteomic data. These findings have implications for future studies pursuant to understanding the proteomic mechanisms underlying dental enamel formation and other associated processes.