An efficient and rapid method for enrichment of lipophilic proteins fromMycobacterium tuberculosisH37Rv for two-dimensional gel electrophoresis

Abstract

Lipophilic proteome profiling is crucial because they have an anticipated role in biological processes and pathogenesis of Mycobacterium tuberculosis. These lipophilic proteins might be used as potential targets for the development of newer diagnostic markers and drug targets due to their association with membranes and drugs. We developed an efficient and rapid method to enrich the lipophilic proteins extraction from M. tuberculosis H37Rv for 2DE. In the extraction of lipophilic proteins, nonionic detergent (Triton X-100) was added in sonication buffer that augmented the solubilization of the proteins at the time of sonication. Enriched whole cell lysate was subjected to direct phase separation using Triton X-114, without the need for preisolation of membranes. In this study, we report that our optimized extraction buffer increased the lipophilic proteins extraction and their improved resolution on 2D gel up to two- to threefolds (quantitatively and qualitatively) as compared to standard extraction buffer. Some proteins were identified by MALDI-TOF/MS.

Cytosolic Proteome Profiling of Aminoglycosides Resistant Mycobacterium tuberculosis Clinical Isolates Using MALDI-TOF/MS

“…Mycobacterial cell lysate was prepared with slight modifications (Brodie et al, 1979; Sharma and Bisht, 2016). Briefly, Cells were suspended in sonication buffer with 1% v/v Triton X-100 and then broken by intermittent sonication at 4°C for 20 min.…”

“…IEF and SDS-PAGE were carried out using the published protocol of “in gel rehydration” with slight modifications (Gorg et al, 2000; Sharma and Bisht, 2016). In brief, IPG strips of pH 4–7 and length 17 cm (Bio-Rad, Hercules, CA, USA) were rehydrated overnight at 20°C with 550 μg proteins.…”

Recent advances in protein analysis by capillary and microchip electrophoresis

“…133 The authors added Triton X-100 in the protein extraction buffer followed by Triton X-114 (for phase separation) directly which eliminated the need for membranes preisolation. The addition of the surfactant to the extraction buffer improved the solubilization of the lipophilic proteins which resulted in 2–3-fold enrichment in MALDI-TOF MS detection of target proteins.…”

Role of Bacterioferritin & Ferritin in M. tuberculosis Pathogenesis and Drug Resistance: A Future Perspective by Interactomic Approach

“…In M. tuberculosis they typically inhibit protein synthesis by interacting with protein translational machinery. Two dimensional gel electrophoresis coupled with mass spectrometry is the best accepted approach for expression proteomics (Lata et al, 2015a,b; Sharma et al, 2015b, 2016b; Sharma and Bisht, 2016). Since last decade a panel of proteomics and bioinformatics studies related to aminoglycosides resistance have been accumulated (Sharma et al, 2010, 2014, 2015b,a, 2016a,b; Kumar et al, 2013; Sharma and Bisht, 2017a,b).…”