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Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS: Preparation of more than 4000 native protein maps

ABSTRACT: Soluble proteins of human bronchial smooth muscle cells (HBSMC) were separated by nondenaturing micro 2DE and a 30 mm X 40 mm area of the CBB-stained slab gel (1.0 mm thick) was cut into 1.1 mm X 1.1 mm squares, then the proteins in the 972 gel pieces (squares) were applied to quantitative LC-MS/MS. Grid-cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC-MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data [Jin, et al. Electrophoresis 2014, 35, 2055-2064, PRIDE Project PXD000852]. Totally 4323 proteins were identified in successfully analyzed 967 squares and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The quantity of the proteins distributed from 3.6 % to 1 X 10-5 % of the total protein quantity in the grid area. Each protein map was characterized by many features, such as the position of quantity peak square, number of detected squares, degree of concentration (focused or dispersed), etc. About 4 percent of the proteins were detected in 100 or more squares, suggesting that they might be ubiquitous and interacting with other proteins. In contrast, many proteins showed more concentrated quantity distribution and the quantity peak positions of 565 proteins with defined degree of concentration were summarized into a quantity peak map. These results for the first time visualized the distribution patterns of cellular proteins on a nondenaturing 2DE gel, which would further provide information on their interactions.

INSTRUMENT(S): Synapt G2-S HDMS

ORGANISM(S): Homo Sapiens (human)

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

SUBMITTER: Ya Jin

LAB HEAD: Ya Jin

PROVIDER: PXD001471 | Pride | 2015-08-10

REPOSITORIES: Pride

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Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS. 1. Preparation of more than 4000 native protein maps.

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

Electrophoresis 20150617 15

Soluble proteins of human bronchial smooth muscle cells (HBSMC) were separated by nondenaturing micro 2DE and a 30 mm × 40 mm area of the CBB-stained slab gel (1.0 mm thick) was cut into 1.1 mm × 1.1 mm squares, then the proteins in the 972 gel pieces (squares) were applied to quantitative LC-MS/MS. Grid-cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC-MS/MS, (iii) reconstruct the protein distribution ...[more]

PMID: 25931155

Publication: 1/2

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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.

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