Peptidomic approaches to study proteolytic activity

Peter J. Lyons, Lloyd D. Fricker

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Peptidomics, the analysis of the peptide content of cells or tissues, can be used to study proteases in several ways. First, nearly all of the peptides detected in cells and tissues are proteolytic fragments of proteins. Analysis of the peptides therefore provides information regarding the proteolytic activities that occurred to generate the observed peptides. The use of quantitative peptidomic approaches allows the comparison of relative peptide levels in two or more different samples, which enables studies examining the consequences of increasing proteolytic activity (by enzyme activation or overexpression) or reducing proteolytic activity (by inhibition, knock down, or knock out). Quantitative peptidomics can also be used to directly test the cleavage specificity of purified proteases. For this, peptides are purified from the tissue or cell line of interest, incubated in the presence of various amounts of protease or in the absence of protease, and then analyzed by the quantitative peptidomics approach. This reveals which peptides are preferred substrates, which are products, and which are not cleaved. Collectively, these studies complement conventional approaches to study proteolytic activity and allow for a more complete understanding of an enzyme's substrate specificity. This unit describes the use of quantitative peptidomics in the analysis of the biological peptidome as well as in the in vitro analysis of peptidase activity.

Original languageEnglish (US)
Article number18.13
JournalCurrent Protocols in Protein Science
Volume1
Issue numberSUPPL.65
DOIs
StatePublished - Aug 2011

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Peptide Hydrolases
Peptides
Tissue
Enzyme Activation
Substrates
Enzymes
Substrate Specificity
Chemical activation
Cells
Cell Line
Proteins

Keywords

  • Mass spectrometry
  • Protease
  • Proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Structural Biology
  • Medicine(all)

Cite this

Peptidomic approaches to study proteolytic activity. / Lyons, Peter J.; Fricker, Lloyd D.

In: Current Protocols in Protein Science, Vol. 1, No. SUPPL.65, 18.13, 08.2011.

Research output: Contribution to journalArticle

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