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Detection of Functional Matrix Metalloproteinases by Zymography
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Detection of in vivo matrix metalloproteinase activity using microdialysis sampling and liquid chromatography/mass

Ying Wang1, Dmitri V Zagorevski, Michelle R Lennartz

  • 1Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 12180, USA.

Analytical Chemistry
|November 13, 2009
PubMed

Insights

A new method quantifies matrix metalloproteinase (MMP) activity using microdialysis. This technique measures MMP-1 and MMP-9 activity in vivo and assesses MMP inhibitor effectiveness.

Area of Science:

  • Biochemistry
  • Pharmacology
  • Analytical Chemistry

Background:

  • Matrix metalloproteinases (MMPs) are enzymes involved in extracellular matrix degradation.
  • Upregulation of MMPs is linked to various pathological conditions.
  • Developing methods to quantify MMP activity in vivo is crucial for disease research.

Purpose of the Study:

  • To develop and validate a quantitative method for measuring MMP-1 and MMP-9 activity using microdialysis.
  • To assess the efficacy of a broad-spectrum MMP inhibitor, GM 6001, in reducing MMP activity in vivo.
  • To enable real-time observation of MMP activity surrounding microdialysis probes.

Main Methods:

  • A liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay was developed to quantify MMP substrates and their N-terminal peptide products.
  • In vitro studies utilized purified human MMP-1 and MMP-9 to optimize the assay and test GM 6001.
  • In vivo studies involved implanting microdialysis probes in rats and infusing MMP substrates with or without GM 6001.

Main Results:

  • The LC/MS/MS method successfully quantified MMP-1 and MMP-9 products.
  • In vitro, 10 microM GM 6001 completely inhibited product formation.
  • In vivo, coinfusion of 50 microM GM 6001 reduced MMP-1 and MMP-9 substrate product formation by 30% and 25%, respectively.

Conclusions:

  • The developed LC/MS/MS microdialysis method is effective for quantifying MMP activity in vivo.
  • Localized delivery of GM 6001 demonstrates significant MMP inhibition in a subcutaneous implantation model.
  • This assay provides a valuable tool for studying MMPs in physiological and pathological contexts.

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