Journal
ELECTROPHORESIS
Volume 36, Issue 15, Pages 1711-1723Publisher
WILEY
DOI: 10.1002/elps.201400573
Keywords
Cellular proteins; LC-MS; MS; Native protein map; Nondenaturing micro 2DE; Quantitative protein-protein interaction
Funding
- National Natural Science Foundation of China [31400798]
- Science and Technology Program of Guangzhou, China [2014J4100022]
- Fundamental Research Funds for the Central Universities [2013ZM0048]
- Guangdong Province Key Laboratory of Fermentation and Enzyme Engineering Open Fund, China [FJ2013002]
- Ministry of Science and Technology of Guangdong Province, China [2012A080204001]
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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 [1]. 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 several features, including the position of quantity peak square, number of detected squares, and degree of concentration (focused or dispersed). About 4% 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 a 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 2D gel.
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