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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cabras T, Iavarone F, Manconi B, Olianas A, Sanna MT, Castagnola M, Messana I (2014) Top-down analytical platforms for the characterization of the human salivary proteome. Bioanalysis 6:563–581. https://doi.org/10.4155/bio.13.349
Amado F, Lobo MJC, Domingues P, Duarte JA, Vitorino R (2010) Salivary peptidomics. Expert Rev Proteomics 7:709–721. https://doi.org/10.1586/epr.10.48
Thomadaki K, Helmerhorst EJ, Tian N, Sun X, Siqueira WL, Walt DR, Oppenheim FG (2011) Whole-saliva proteolysis and its impact on salivary diagnostics. J Dent Res 90:1325–1330
Vitorino R, Barros A, Caseiro A, Domingues P, Duarte J, Amado F (2009) Towards defining the whole salivary peptidome. Proteomics Clin Appl 3:528–540. https://doi.org/10.1002/prca.200800183
Lucchi G, Chambon C, Truntzer C, Pecqueur D, Ducoroy P, Schwartz C, Nicklaus S, Morzel M (2009) Mass-spectrometry based characterisation of infant whole saliva peptidome. Int J Pept Res Ther 15:177–185. https://doi.org/10.1007/s10989-009-9167-2
Sun X, Salih E, Oppenheim FG, Helmerhorst EJ (2009) Activity-based mass spectrometric characterization of proteases and inhibitors in human saliva. Proteomics Clin Appl 3:810–820. https://doi.org/10.1002/prca.200800242
Wiśniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6:359–362
Manza LL, Stamer SL, Ham A-JL, Codreanu SG, Liebler DC (2005) Sample preparation and digestion for proteomic analyses using spin filters. Proteomics 5:1742–1745. https://doi.org/10.1002/pmic.200401063
Trindade F, Amado F, Gomes PS, Vitorino R (2015) endoProteoFASP: a novel FASP approach to profile salivary peptidome and disclose salivary proteases. Talanta 132:486–493. https://doi.org/10.1016/j.talanta.2014.09.026
Trindade F, Amado F, Oliveira-Silva RP, Daniel-da-Silva AL, Ferreira R, Klein J, Faria-Almeida R, Gomes PS, Vitorino R (2015) Toward the definition of a peptidome signature and protease profile in chronic periodontitis. Proteomics Clin Appl 9:917–927. https://doi.org/10.1002/prca.201400191
Klein J, Eales J, Zürbig P, Vlahou A, Mischak H, Stevens R (2013) Proteasix: a tool for automated and large-scale prediction of proteases involved in naturally occurring peptide generation. Proteomics 13:1077–1082. https://doi.org/10.1002/pmic.201200493
Arguello Casteleiro M, Klein J, Stevens R (2016) The proteasix ontology. J Biomed Semantics 7:1–7. https://doi.org/10.1186/s13326-016-0078-9
Hölttä M, Zetterberg H, Mirgorodskaya E, Mattsson N, Blennow K, Gobom J (2012) Peptidome analysis of cerebrospinal fluid by LC-MALDI MS. PLoS One 7:e42555. https://doi.org/10.1371/journal.pone.0042555
Bery A, Leung F, Smith CR, Diamandis EP, Kulasingam V (2014) Deciphering the ovarian cancer ascites fluid peptidome. Clin Proteomics 11:1–9. https://doi.org/10.1186/1559-0275-11-13
Cova M, Oliveira-Silva R, Ferreira JA, Ferreira R, Amado F, Daniel-da-Silva AL, Vitorino R (2015) Glycoprotein enrichment method using a selective magnetic nano-probe platform (mnp) functionalized with lectins BT - clinical proteomics: methods and protocols. In: Vlahou A, Makridakis M (eds) . Springer, New York, pp 83–100
Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A, Salazar GA, Tate J, Bateman A (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44:D279–D285. https://doi.org/10.1093/nar/gkv1344
Finn RD, Mistry J, Schuster-Böckler B, Griffiths-Jones S, Hollich V, Lassmann T, Moxon S, Marshall M, Khanna A, Durbin R, Eddy SR, Sonnhammer ELL, Bateman A (2006) Pfam: clans, web tools and services. Nucleic Acids Res 34:D247–D251. https://doi.org/10.1093/nar/gkj149
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7537-2_19
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7536-5
Online ISBN: 978-1-4939-7537-2
eBook Packages: Springer Protocols