Dataset Information

Mycobacterium tuberculosis culture filtrate LC-MS/MS

ABSTRACT: Triplicate culture filtrate samples of normal (log phase) and PBS cultured (starvation) M. tuberculosis were separated by SDS-PAGE, and 10 bands were excised from each lane after staining with Coomassie R-250. In-gel trypsin digestion was performed on gel bands followed by nanoLC. Eluting peptides were then analyzed on an LTQ FT instrument, and the resulting raw files were searched with Sequest (part of Proteome Discoverer) against an M. tuberculosis H37Rv database (downloaded from the Sanger Centre). The final combined dataset comprised 12,399 unique peptides derived from 1,362 proteins, each identified by a minimum of two distinct peptides.

REANALYSED by: PAe003971PAe003970

INSTRUMENT(S): LTQ FT

ORGANISM(S): Mycobacterium Tuberculosis H37rv

SUBMITTER: Ida Rosenkrands

PROVIDER: PXD000111 | Pride | 2013-01-24

REPOSITORIES: Pride

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Publications

Proteomic profiling of Mycobacterium tuberculosis identifies nutrient-starvation-responsive toxin-antitoxin systems.

Albrethsen Jakob J Agner Jeppe J Piersma Sander R SR Højrup Peter P Pham Thang V TV Weldingh Karin K Jimenez Connie R CR Andersen Peter P Rosenkrands Ida I

Molecular & cellular proteomics : MCP 20130123 5

In order to successfully enter the latent stage, Mycobacterium tuberculosis must adapt to conditions such as nutrient limitation and hypoxia. In vitro models that mimic latent infection are valuable tools for describing the changes in metabolism that occur when the bacterium exists in a non-growing form. We used two complementary proteomic approaches, label-free LC-MS/MS analysis and two-dimensional difference gel electrophoresis, to determine the proteome profile of extracellular proteins from ...[more]

PMID: 23345537

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Project description:Proteomic analysis by combining PAGE separation, gel slicing and slice-by-slice LC-MS/MS has been frequently reported in recent years. Since the MS analysis would provide identities and quantities of the proteins along the whole lane, visualization by dye staining could be skipped to save time and labor and also in some reports assumed to improve MS identification and sensitivity. In this work, we examined the effect of CBB R-250 staining on the performance of the method and the results showed actually better results were obtained with CBB staining than without. A primary examination was firstly performed with gel bands of purified proteins, in which eight protein bands were excised, each from both the CBB-stained and unstained gel parts, and then analyzed by in-gel digestion and quantitative LC-MS/MS. Almost all the proteins were detected with higher sequence coverages and quantities from the stained gel bands than from the unstained. Then a proteomic sample of rat heart soluble proteins was examined for the complete workflow. The sample was firstly separated by nondenaturing PAGE and the gel was divided to two halves, with one CBB-stained and the other unstained. Laboratory-made tools were used to simultaneously cut duplicate lanes from each gel half and then slice each lane into about 39 pieces of the same size (1.1 mm 椋?1.1 mm 椋?1 mm thick). All the gel square pieces were analyzed in standardized procedures of in-gel digestion, peptide extraction and label-free quantitative LC-MS/MS. The results showed an average of 1434 proteins were detected in CBB-stained lanes, 40% higher than the 1013 in unstained lanes. When proteins detected in both conditions were compared, most of them were detected in higher quantities and in more gel squares with CBB staining. The comparison was also performed for SDS-PAGE and similarly advantageous results were obtained from the CBB-stained lanes, in both the detected numbers of proteins and peptides and the detected quantities and square numbers of individual proteins. The data also showed the proteins with lower molecular masses (e.g. < 30 kDa) were more benefited by the staining, probably because the dye binding helped to retain the proteins in gel matrix. In short, though dye staining is no longer a requisite when PAGE separation is followed by whole-gel LC-MS/MS analysis, CBB staining is still recommended for the better detection in proteomic analysis. All the raw and search files of the LC-MS/MS analysis, for (8*4=) 32 gel squares of HMW and LMW markers and (39*4+41*4=) 320 squares of rat heart soluble proteins (totally 704 files), as well as the gel patterns (2 files) and the summaries of the protein-level search results (3 files), are deposited in this project.

Dataset Information

Mycobacterium tuberculosis culture filtrate LC-MS/MS

Publications

Proteomic profiling of Mycobacterium tuberculosis identifies nutrient-starvation-responsive toxin-antitoxin systems.

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