Multidimensional analysis of protease substrates and their cellular origins in mixed secretomes from multiple cell types

Pascal Schlage, Ulrich auf dem Keller*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

Abstract

Although extracellular proteases are confronted with substrate proteins expressed by multiple cell types in vivo, in most protease substrate discovery approaches, the test protease is exposed to a test proteome (secretome) derived only from a single cell type. This limits the potential substrate space and prohibits the formation of protein complexes constituted of components derived from multiple cellular origins. Mixing of secretomes collected from multiple cell types addresses this issue, but information on the cellular origin of a substrate protein is lost. Here, we describe a protocol and the corresponding data analysis workflow for a multidimensional substrate discovery approach termed SILAC-iTRAQ-TAILS that is based on hyperplexed terminal amine isotopic labeling of substrates (TAILS), allowing identification of substrates and concomitant assignment to cellular origins in mixed secretomes within the same experiment.

Original languageEnglish
Title of host publicationProtein Terminal Profiling
Number of pages14
Volume1574
PublisherHumana Press
Publication date2017
Pages91-104
Chapter7
ISBN (Print)978-1-4939-6849-7
ISBN (Electronic)978-1-4939-6850-3
DOIs
Publication statusPublished - 2017
Externally publishedYes
SeriesMethods in Molecular Biology
Volume1574
ISSN1064-3745

Keywords

  • iTRAQ
  • Protease substrate
  • Secretome
  • SILAC
  • TAILS

Cite this

Schlage, P., & auf dem Keller, U. (2017). Multidimensional analysis of protease substrates and their cellular origins in mixed secretomes from multiple cell types. In Protein Terminal Profiling (Vol. 1574, pp. 91-104). Humana Press. Methods in Molecular Biology, Vol.. 1574 https://doi.org/10.1007/978-1-4939-6850-3_7
Schlage, Pascal ; auf dem Keller, Ulrich. / Multidimensional analysis of protease substrates and their cellular origins in mixed secretomes from multiple cell types. Protein Terminal Profiling . Vol. 1574 Humana Press, 2017. pp. 91-104 (Methods in Molecular Biology, Vol. 1574).
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Schlage, P & auf dem Keller, U 2017, Multidimensional analysis of protease substrates and their cellular origins in mixed secretomes from multiple cell types. in Protein Terminal Profiling . vol. 1574, Humana Press, Methods in Molecular Biology, vol. 1574, pp. 91-104. https://doi.org/10.1007/978-1-4939-6850-3_7

Multidimensional analysis of protease substrates and their cellular origins in mixed secretomes from multiple cell types. / Schlage, Pascal; auf dem Keller, Ulrich.

Protein Terminal Profiling . Vol. 1574 Humana Press, 2017. p. 91-104 (Methods in Molecular Biology, Vol. 1574).

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

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AB - Although extracellular proteases are confronted with substrate proteins expressed by multiple cell types in vivo, in most protease substrate discovery approaches, the test protease is exposed to a test proteome (secretome) derived only from a single cell type. This limits the potential substrate space and prohibits the formation of protein complexes constituted of components derived from multiple cellular origins. Mixing of secretomes collected from multiple cell types addresses this issue, but information on the cellular origin of a substrate protein is lost. Here, we describe a protocol and the corresponding data analysis workflow for a multidimensional substrate discovery approach termed SILAC-iTRAQ-TAILS that is based on hyperplexed terminal amine isotopic labeling of substrates (TAILS), allowing identification of substrates and concomitant assignment to cellular origins in mixed secretomes within the same experiment.

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Schlage P, auf dem Keller U. Multidimensional analysis of protease substrates and their cellular origins in mixed secretomes from multiple cell types. In Protein Terminal Profiling . Vol. 1574. Humana Press. 2017. p. 91-104. (Methods in Molecular Biology, Vol. 1574). https://doi.org/10.1007/978-1-4939-6850-3_7