Nano-scale proteomics approach using two-dimensional fibrin zymography combined with fluorescent SYPRO ruby dye.
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).
Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea.
Nack-Shick
NS
Choi
Ki-Hyun
KH
Yoo
Kab-Seog
KS
Yoon
Pil Jae
PJ
Maeng
Seung-Ho
SH
Kim
D000818:Animals; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D002417:Cattle; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D005063:Europium / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D036103:Nanotechnology; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; C436863:SYPRO Rose Plus; D005063:Europium; D005337:Fibrin; D010450:Endopeptidases