Mapping the N-Terminome in Tissue Biopsies by PCT-TAILS

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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.
Original languageEnglish
Title of host publicationADAMTS Proteases
Number of pages12
Volume2043
Publication date2019
Pages285-296
Chapter24
ISBN (Print)978-1-4939-9697-1
ISBN (Electronic)978-1-4939-9698-8
DOIs
Publication statusPublished - 2019
SeriesMethods in Molecular Biology
ISSN1064-3745

Keywords

  • Proteolysis
  • Degradomics
  • Protease
  • Pressure cycling technology
  • PCT-TAILS
  • Tissue biopsies
  • Proteomics

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