Abstract
Proteases play pivotal roles in multiple biological processes in all living organisms and are tightly regulated under normal conditions, but alterations in the proteolytic system and uncontrolled protease activity result in multiple pathological conditions. A disease will most often be defined by an ensemble of cleavage events—a proteolytic signature, thus the system-wide study of protease substrates has gained significant attention and identification of disease specific clusters of protease substrates holds great promise as targets for diagnostics and therapy.
In this chapter we describe a method that enables fast and reproducible analysis of protease substrates and proteolytic products in an amount of tissue less than the quantity obtained by a standard biopsy. The method combines tissue disruption and protein extraction by pressure cycling technology (PCT), N-terminal enrichment by tandem mass tag (TMT)-terminal amine isotopic labeling of substrates (TAILS), peptide analysis by mass spectrometry (MS), and a general pipeline for interpretation of the data.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
LĂłpez-OtĂn C, Bond JS (2008) Proteases: multifunctional enzymes in life and disease. J Biol Chem 283:30433–30437
Pérez-Silva JG, Español Y, Velasco G et al (2016) The degradome database: expanding roles of mammalian proteases in life and disease. Nucleic Acids Res 44:D351–D355
Fortelny N, Cox JH, Kappelhoff R et al (2014) Network analyses reveal pervasive functional regulation between proteases in the human protease web. PLoS Biol 12:e1001869
McCarty SM, Percival SL (2013) Proteases and delayed wound healing. Adv Wound Care 2:438–447
Breznik B, Motaln H, Lah Turnšek T (2017) Proteases and cytokines as mediators of interactions between cancer and stromal cells in tumours. Biol Chem 398:709–719
De Stefano ME, Herrero MT (2017) The multifaceted role of metalloproteinases in physiological and pathological conditions in embryonic and adult brains. Prog Neurobiol 155:36–56
Weiss-Sadan T, Gotsman I, Blum G (2017) Cysteine proteases in atherosclerosis. FEBS J 284:1455–1472
Edgington-Mitchell LE (2016) Pathophysiological roles of proteases in gastrointestinal disease. Am J Physiol Liver Physiol 310:G234–G239
Lobmann R, Ambrosch A, Schultz G et al (2002) Expression of matrix-metalloproteinases and their inhibitors in the wounds of diabetic and non-diabetic patients. Diabetologia 45:1011–1016
auf dem Keller U, Prudova A, Eckhard U et al (2013) Systems-level analysis of proteolytic events in increased vascular permeability and complement activation in skin inflammation. Sci Signal. https://doi.org/10.1126/scisignal.2003512
Broder C, Arnold P, Vadon-Le Goff S et al (2013) Metalloproteases meprin and meprin are C- and N-procollagen proteinases important for collagen assembly and tensile strength. Proc Natl Acad Sci 110:14219–14224
Huesgen PF, Lange PF, Overall CM (2014) Ensembles of protein termini and specific proteolytic signatures as candidate biomarkers of disease. Proteomics Clin Appl 8:338–350
Guo T, Kouvonen P, Koh CC et al (2015) Rapid mass spectrometric conversion of tissue biopsy samples into permanent quantitative digital proteome maps. Nat Med 21:407–413
Goldberg S (2015) Mechanical/physical methods of cell distribution and tissue homogenization. Methods Mol Biol 1295:1–20
Balny C, Masson P, Heremans K (2002) High pressure effects on biological macromolecules: from structural changes to alteration of cellular processes. Biochim Biophys Acta 1595:3–10
Gross V, Carlson G, Kwan AT et al (2008) Tissue fractionation by hydrostatic pressure cycling technology: the unified sample preparation technique for systems biology studies. J Biomol Tech 19:189–199
Ringham H, Bell RL, Smejkal GB et al (2007) Application of pressure cycling technology to tissue sample preparation for 2-DE. Electrophoresis 28:1022–1024
Zhu Y, Guo T (2017) High-throughput proteomic analysis of fresh-frozen biopsy tissue samples using pressure cycling technology coupled with SWATH mass spectrometry. Methods Mol Biol 1788:279–287
Shao S, Guo T, Gross V et al (2016) Reproducible tissue homogenization and protein extraction for quantitative proteomics using micropestle-assisted pressure-cycling technology. J Proteome Res 15:1821–1829
Kleifeld O, Doucet A, auf dem Keller U et al (2010) Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat Biotechnol 28:281–288
Schlage P, Egli FE, Nanni P et al (2014) Time-resolved analysis of the matrix metalloproteinase 10 substrate degradome. Mol Cell Proteomics 13:580–593
Sabino F, Hermes O, Egli FE et al (2015) In vivo assessment of protease dynamics in cutaneous wound healing by degradomics analysis of porcine wound exudates. Mol Cell Proteomics 14:354–370
Schlage P, Kockmann T, Sabino F et al (2015) Matrix metalloproteinase 10 degradomics in keratinocytes and epidermal tissue identifies bioactive substrates with pleiotropic functions. Mol Cell Proteomics 14:3234–3246
Sabino F, Egli FE, Savickas S et al (2018) Comparative degradomics of porcine and human wound exudates unravels biomarker candidates for assessment of wound healing progression in trauma patients. J Invest Dermatol 138:413–422
Kleifeld O, Doucet A, Prudova A et al (2011) Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates. Nat Protoc 6:1578–1611
auf dem Keller U, Overall CM (2012) CLIPPER: an add-on to the trans-proteomic pipeline for the automated analysis of TAILS N-terminomics data. Biol Chem 393:1477–1483
Schlage P, Egli FE, auf dem Keller U (2017) Time-resolved analysis of matrix metalloproteinase substrates in complex samples. Methods Mol Biol 1579:185–198
Acknowledgments
We thank Erwin Schoof and Lene Holberg Blicher for continuous support in operating the DTU Proteomics Core. This work was supported by a Novo Nordisk Foundation Young Investigator Award (NNF16OC0020670)Â and a grant from the Swiss National Science Foundation (31003A_163216) to U.a.d.K.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Bundgaard, L., Savickas, S., auf dem Keller, U. (2020). Mapping the N-Terminome in Tissue Biopsies by PCT-TAILS. In: Apte, S. (eds) ADAMTS Proteases. Methods in Molecular Biology, vol 2043. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9698-8_24
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9698-8_24
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9697-1
Online ISBN: 978-1-4939-9698-8
eBook Packages: Springer Protocols