Salivary Protease Activity In Experimental Gingivitis Revealed By ENDO-FASP Approach.

Objectives: Gingivitis is ubiquitous and a prerequisite for periodontitis to develop. The 21-day experimental gingivitis model allows investigation of the induction and resolution of gingival inflammation. Saliva bathes the oral cavity continuously and may change in composition in response to inflammation. the objective was to assess the impact of experimental gingivitis upon saliva protease activity by using the ENDO-FASP technique.

Methods: 9 healthy volunteers enrolled on a 21 day experimental gingivitis study. Plaque index (PI) and gingival index (GI) were recorded at days 0 and 21; and 14 days following resumption of normal hygiene (day 35). Stimulated saliva was collected for 5min at each appointment on to ice and stored at -80°C until use. Protein composition and protease activity were measured by the ENDO-FASP technique whereby the endogenous proteases were allowed to degrade saliva proteins. The resulting peptides were then detected using one dimensional liquid chromatography tandem mass spectrometry. Data were analysed by Proteome Discoverer and Proteasix softwares. Protease levels were assessed for differences between stages of the clinical protocol using linear mixed model regressions.

Results: PI and GI at test sites demonstrated successful induction and subsequent resolution of inflammation. 783 proteins were found in total with an average of 162 proteins per sample. 61 proteins were found in all samples. The approach used allowed for detection of non-tryptic peptides and discovery of highly abundant saliva proteins not normally detected by traditional proteomic techniques, eg Proline-rich proteins. Analysis of protease activity predicted the activity of 36 proteases in saliva. After correction for multiple testing, the models showed that the relative activity of most proteases did not appear to change across the course of the experiment.
Conclusions: While computationally hungry, the use of non-traditional proteomic methods allowed for discovery of highly abundant saliva proteins usually missed. Analysis of protease activity suggests tight regulation.