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Related Concept Videos

Tagging and Fusion Proteins01:24

Tagging and Fusion Proteins

Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...

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Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation
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Published on: January 16, 2019

Multiplexed protease assays using element-tagged substrates.

Urja S Lathia1, Olga Ornatsky, Vladimir Baranov

  • 1Department of Chemistry, University of Toronto, Ontario, Canada.

Analytical Biochemistry
|September 21, 2010
PubMed
Summary
This summary is machine-generated.

Inductively coupled plasma-mass spectrometry (ICP-MS) enables multiplexed protease assays. This study demonstrates a quadruplex assay for cysteine and metalloproteases, showing a simple, reproducible method with high accuracy.

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Proteomics

Background:

  • Inductively coupled plasma-mass spectrometry (ICP-MS) offers high sensitivity, resolution, and reliability, making it suitable for multiplexed assays.
  • Protease activity is crucial in various biological processes and disease states, necessitating robust assay development.

Purpose of the Study:

  • To demonstrate the potential of ICP-MS-based assays for multiplexed protease activity detection.
  • To develop and validate a quadruplex assay for simultaneous quantification of specific cysteine and metalloproteases.

Main Methods:

  • Synthesis of four orthogonal peptide substrates, each functionalized with a biotin tag and a DTPA-lanthanide complex.
  • Development of an ICP-MS based assay for simultaneous detection of cleavage events on the four substrates.
  • Validation of the multiplex assay against single-analyte assays.

Main Results:

  • Successful development of a quadruplex ICP-MS assay for cysteine proteases (calpain-1, caspase-3) and metalloproteases (MMP-9, ADAM10).
  • Demonstrated high correlation between single and multiplex assay formats, indicating assay reliability and accuracy.
  • Highlighted the simplicity and reproducibility of the ICP-MS based multiplexing approach.

Conclusions:

  • ICP-MS is a powerful technique for developing sensitive and reproducible multiplexed protease assays.
  • The developed quadruplex assay provides a valuable tool for simultaneous analysis of multiple protease activities.
  • This approach has broad applicability in biological research and diagnostics.