Quantitative proteomics in rat saliva stimulated with pilocarpine and isoprenaline

Objective: Saliva is increasingly being recognized as a convenient and informative reservoir of proteins that could serve as indicators of various diseases. As the literature remains taciturn with regard to saliva collection methods in rodents, our objective was to provide the protocol for a comprehensive quantitative proteomic assessment of stimulated rat saliva. Design: We applied the next-generation proteomic methodology (directDIA) to compare qualitatively and quantitatively stimulated rat saliva specimens obtained from pilocarpine alone and pilocarpine in combination with isoprenaline. Results: Collectively, we identified 581 protein groups with high confidence across all samples included in the analysis, with the dynamic range of the identifications estimated to cover 5 orders of magnitude difference between the most abundant and least abundant salivary proteins. Our data evidenced that pilocarpine-stimulated saliva collection showed a trend towards more protein groups identified; however, quantitative reproducibility was preferable after dual stimulation. Conclusions: The main advantage of the double stimulation strategy is the quantitative stability of the salivary proteome, crucial for quantitative salivaomic experiments. We postulate that the latter in combination with the depth of proteome analysis provided by the directDIA technique constitutes a novel analytical tool in research studies designed to unravel the saliva protein composition and its changes in vivo.