Two dimensional gel electrophoresis (2-DE) for high-throughput proteome analyses of Mycoplasma bovis.

Abstract

Expression proteomics approaches do not only directly confirm protein coding genes of sequenced genomes but also facilitate resolution of minute qualitative protein differences and improve the quality of genome annotation. Despite development of many tools, use of 2-DE coupled with MS in proteomics is not uncommon. With an accelerated trend of genome sequencing of microorganisms, proteome analysis of animal pathogens with 2-DE has gained more attention in the last decade. Therefore, in this study primarily the protein extraction, sample preparation and loading, IPG strip rehydration, IEF, and SDS-PAGE conditions were improved for high throughput resolution and reproducible 2-DE map of proteins of Mycoplasma bovis HB0801 (M. bovis HB0801- Chinese Strain), a pneumonia pathogen in feedlot cattle, and its attenuated strains. Literally, higher amount of proteins was extracted exploiting the French pressure cell coupled with TCA precipitation when compared to the sonication method. Total protein concentration was determined using a 2D quant Kit. About 330-380 μg TCA treated protein sample, solubilized in calibrated rehydration solution, loaded on 17 cm IPG gel strip (pH 3-10 NL) followed by active rehydration at 50V and isoelectric focusing at final 10 000 Volt (33 uA/gel strip) for 80kVh had revealed well resolved proteins spots on 10% gel stained by CBB R250 (0.15%), representing 83-89% of the total protein coding genes of M. bovis HB0801, estimated by PD Quest (Bio-Rad, USA). Conclusively, this effort attempted to provide more precise 2-DE platform and suitable conditions, after extensive calibration, for future comprehensive proteome and immunoproteome analyses and future research on the elucidation of factors related to pathogenesis of M. bovis in cattle.