Dataset Information

Analysis of proteins in supernatant and precipitate fractions of human bronchial smooth muscle cells by 1D SDS-PAGE and quantitative LC-MS/MS

ABSTRACT: Supernatant and precipitate protein fractions of human bronchial smooth muscle cells (HBSMC) after sonication and centrifugation were analyzed using 1D SDS-PAGE. The gel lanes were cube-cut into thirty-three 1.1 mm x 1.1 mm x 1 mm (gel thickness) squares and each gel piece was subjected to in-gel digestion and quantitative LC-MS/MS. Totally 2552 proteins were assigned from the lane of supernatant and 2614 protein species from the lane of precipitate. The results are being further analyzed, e.g. 1) for the comparison between the proteins species residing in the two fractions, and 2) for correlation with the nondenaturing 2DE-grid cut-quantitative LC-MS/MS results (Jin, et al. Electrophoresis 2015, 36, 1711-1723, Jin, et al. Electrophoresis 2015, 1991-2001, PRIDE project PXD001471).

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Bronchial Smooth Muscle Cell

SUBMITTER: Ya Jin

LAB HEAD: Ya Jin

PROVIDER: PXD010036 | Pride | 2019-01-03

REPOSITORIES: Pride

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Publications

Comparison of the performance of 1D SDS-PAGE with nondenaturing 2DE on the analysis of proteins from human bronchial smooth muscle cells using quantitative LC-MS/MS.

Jin Ya Y Zhang Jun J Manabe Takashi T Tan Wen W

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 20181224

The supernatant and precipitate protein fractions from human bronchial smooth muscle cells (HBSMC) were analyzed using 1D SDS-PAGE-LC-MS/MS and the performance of the method was compared with that of nondenaturing 2DE-LC-MS/MS applied to the supernatant fraction, the methods and the results have been reported previously. The 1D gel lanes were cut into pieces of the same size, in order to enable the reproduction of digital 1D images of the individual MS-assigned proteins. When the obtained inform ...[more]

PMID: 30599360

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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

Analysis of proteins in supernatant and precipitate fractions of human bronchial smooth muscle cells by 1D SDS-PAGE and quantitative LC-MS/MS

Publications

Comparison of the performance of 1D SDS-PAGE with nondenaturing 2DE on the analysis of proteins from human bronchial smooth muscle cells using quantitative LC-MS/MS.

Similar Datasets

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