Abstract
In the quest to fully comprehend the proteolytic events leading to the generation of the salivary peptidome, we have developed a method for the sequential elution of salivary peptides throughout progressive endogenous proteolysis. By screening the time-dependent changes in the salivary peptidome we can predict the activity pattern of salivary proteases responsible for such peptide fingerprint and identify susceptible protein targets. Herein, we describe a step-by-step tutorial based on a filter-aided sample preparation (FASP) method, taking advantage of the endogenous salivary proteases armamentarium (endoProteoFASP), to produce new peptides from the salivary proteins, adding to those present in the sample at the time of collection. In this protocol, the different sets of peptides retrieved after sample elution are identified following a liquid chromatography-tandem mass spectrometry approach. The likelihood of a large set of endogenous proteases (collected from several public sources) to be responsible for the generation of such peptides can be predicted by the analysis of the cleavage site specificity by Proteasix (http://proteasix.cs.man.ac.uk/) algorithm. The attained peptidome-protease profile can be useful to elucidate the peptidome dynamics and the proteolytic events underpinning pathophysiological phenomena taking place locally within the oral cavity. This may help clinicians to diagnose oral pathologies and develop preventive therapeutic plans.
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Acknowledgments
This work was supported by the Portuguese Foundation for Science and Technology (FCT) through UnIC, iBiMED, QOPNA research units (UID/IC/00051/2013, UID/BIM/04501/2013, PEst-C/QUI/UI0062/2013) and by project DOCnet (NORTE-01-0145-FEDER-000003), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Rui Vitorino and Fábio Trindade are supported by individual fellowship grants (IF/00286/2015 and SFRH/BD/111633/2015, respectively).
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Trindade, F. et al. (2018). EndoProteoFASP as a Tool to Unveil the Peptidome-Protease Profile: Application to Salivary Diagnostics. In: Schrader, M., Fricker, L. (eds) Peptidomics. Methods in Molecular Biology, vol 1719. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7537-2_19
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DOI: https://doi.org/10.1007/978-1-4939-7537-2_19
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