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).
Choi
Nack-Shick
NS
Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea.
Yoo
Ki-Hyun
KH
Yoon
Kab-Seog
KS
Maeng
Pil Jae
PJ
Kim
Seung-Ho
SH
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