Nano-scale proteomics approach using two-dimensional fibrin zymography combined with fluorescent SYPRO ruby dye.

Abstract

In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE). Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp. proteases. Namely, complex protein mixtures from Bacillus sp. DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel. The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis. Five intracellular fibrinolytic enzymes of Bacillus sp. DJ-4 were detected through 2-D FZ. The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID-TOF MS. Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis. Also, the extracellular proteases of Bacillus sp. DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established. This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).

• Authors: Nack-Shick Choi, Ki-Hyun Yoo, Kab-Seog Yoon, Pil Jae Maeng, Seung-Ho Kim
• Journal: Journal of biochemistry and molecular biology (J Biochem Mol Biol) Vol. 37 Issue 3 Pg. 298-303 (May 31 2004) ISSN: 1225-8687 [Print] Korea (South)
• PMID: 15469710 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bacterial Proteins
• Fluorescent Dyes
• SYPRO Rose Plus
• Europium
• Fibrin
• Endopeptidases

Topics

• Animals
• Bacillus (chemistry, enzymology)
• Bacterial Proteins (chemistry, metabolism)
• Biological Assay (methods)
• Cattle
• Endopeptidases (chemistry, metabolism)
• Europium (metabolism)
• Fibrin (metabolism)
• Fluorescent Dyes (metabolism)
• Isoelectric Focusing
• Molecular Sequence Data
• Nanotechnology
• Proteomics (methods)
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization