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.