Protein extraction from Mycobacterium avium subsp. paratuberculosis: Comparison of methods for analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis, native PAGE and surface enhanced laser desorption/ionization time of flight mass spectrometry.
Mycobacterium paratuberculosis causes Johne's disease, a chronic bowel disease in ruminants worldwide and is currently incurable. This study was conducted to compare methods for examining the proteome of M. paratuberculosis. SDS-PAGE, native PAGE and SELDI-TOF-MS were compared and the efficacy of various lysis buffers was assessed. Chaotropic agents (Urea CHAPS and potassium thiocyanate) and non-ionic detergent (Tween20 and Triton X-100) extracts were compared on three different ProteinChip surfaces along with two energy absorbing molecules (EAM): EAM-1 proprietary formulation and sinapinic acid (Ciphergen). Urea CHAPS was efficient for extraction of proteins and their detection on all the ProteinChip surfaces. However, potassium thiocyanate was the most effective buffer, leading to detection of the greatest number of protein peaks on the immobilized metal affinity chromatography (IMAC) surface. Sinapinic acid was more efficient than the EAM-1 proprietary formulation and resulted in additional peaks with higher intensity for both the low and the medium molecular weight range proteins. Intra-chip and inter-chip coefficient of variation for mass/charge varied from 0.01% to 0.07% and 0.00% to 0.08%, respectively. SELDI-TOF-MS was an efficient tool for the protein profiling of M. paratuberculosis and will be useful for investigation of novel proteins, although SDS-PAGE/2D gel electrophoresis is recommended for study of high molecular weight species. All buffers were suitable for protein extraction for SDS-PAGE, while Tween20 was best for native PAGE.
Gumber
Sanjeev
S
Faculty of Veterinary Science, The University of Sydney, Private Bag 3, Camden 2570, NSW Australia.
Taylor
Deborah L
DL
Whittington
Richard J
RJ
D001426:Bacterial Proteins / Q000302:isolation & purification*; D002021:Buffers; D002793:Cholic Acids / Q000737:chemistry; D003373:Coumaric Acids; D004591:Electrophoresis, Polyacrylamide Gel; D016927:Mycobacterium avium subsp. paratuberculosis / Q000737:chemistry* / Q000378:metabolism; D017830:Octoxynol / Q000737:chemistry; D011136:Polysorbates / Q000737:chemistry; D040081:Protein Array Analysis; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013861:Thiocyanates / Q000737:chemistry
D001426:Bacterial Proteins; D002021:Buffers; D002793:Cholic Acids; D003373:Coumaric Acids; D011136:Polysorbates; D013861:Thiocyanates; C073734:sinapinic acid; D017830:Octoxynol; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C009941:potassium thiocyanate