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Methods Mol Biol. 2018;1719:293-310. doi: 10.1007/978-1-4939-7537-2_19.

EndoProteoFASP as a Tool to Unveil the Peptidome-Protease Profile: Application to Salivary Diagnostics.

Author information

1
Department of Medical Sciences, iBiMED, Institute for Biomedicine, University of Aveiro, Aveiro, Portugal. fabiotrindade@ua.pt.
2
Departamento de Cirurgia e Fisiologia, Faculdade de Medicina, UnIC, Unidade de Investigação Cardiovascular, Universidade do Porto, Porto, Portugal. fabiotrindade@ua.pt.
3
Departamento de Cirurgia e Fisiologia, Faculdade de Medicina, UnIC, Unidade de Investigação Cardiovascular, Universidade do Porto, Porto, Portugal.
4
Faculty of Dental Medicine, Laboratory for Bone Metabolism and Regeneration, University of Porto, Porto, Portugal.
5
REQUIMTE/LAQV, University of Porto, Porto, Portugal.
6
INSERM U1048, Institute of Cardiovascular and Metabolic Disease, Toulouse, France.
7
Université Toulouse III Paul-Sabatier, Toulouse, France.
8
Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal.
9
Department of Medical Sciences, iBiMED, Institute for Biomedicine, University of Aveiro, Aveiro, Portugal.

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.

KEYWORDS:

EndoProteoFASP; FASP; Peptidome; Peptidomics; Protease; Proteasix; Saliva

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