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Native proteomic analysis for cellular proteins by nondenaturing 2de and wholegel MS mapping

Native proteomic analysis for cellular proteins by nondenatu...

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2018年中国质谱学术大会（CMSC2018）

作者： Ya Jin Jun Zhang Takashi Manabe Wen Tan Institute of Biomedical and Pharmaceutical Sciences Guangdong University of Technology dermatology Hospital Southern Medical University Faculty of Science Ehime University

nondenaturing 2 de is a technique of almost the highest resolution for separating proteomic samples under native conditions. The retained structures and interactions of proteins in the gel, if could be unveiled, would greatly facilitate understanding on composition and functions of the proteomes. However, the present detection methods for the 2 de-separated proteins could not have satisfying performance for this purpose. For example, the methods of dye staining followed by spot picking and MS identification is often limited in sensitivity and ability to differentiate overlapped proteins, while Western blotting relies on availability of antibodies and has very low throughput. Here we report on our method development for MS mapping of nondenaturing 2 de-separated cellular proteins and the therefore enabled large-scale interaction analysis. Soluble proteins of human bronchial smooth muscle cells(HBSMC) were separated by nondenaturing micro 2 de and a 30 mm × 40 mm area was cut into 1.1 mm × 1.1 mm squares, then the 972 square gel pieces were analyzed with standardized procedures of in-gel digestion and quantitative LC-MS/MS. Totally 4323 proteins were identified and the quantity distribution of each was reconstructed as a color density pattern(a native protein map). As expected, many proteins showed distributions not restricted in a single focused area, suggesting protein interactions were widely present in the cellular protein system. Since the proteins comprising a protein complex are expected to migrate together under the nondenaturing separation and result in similar maps, we developed an algorithm and corresponding computer programs to calculate an "overlap factor" of each protein map against each of the other protein maps. Maps of 2328 proteins that were detected in 3 or more squares were subjected to this evaluation, resulting in a 2328 × 2328 matrix of overlap factors. From the matrix, 431 protein pairs showed overlap factors over 0.65 from both protein sides,

关键词： native cellular proteomics MS mapping protein interactions nondenaturing 2de LC-MS/MS

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Profiling of human HDL and LDL associated proteins by native 2de-MS mapping

Profiling of human HDL and LDL associated proteins by native...

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2018年中国质谱学术大会（CMSC2018）

作者： Ya Jin Shujie Bu Jin Chen Takashi Manabe Wen Tan Institute of Biomedical and Pharmaceutical Sciences Guangdong University of Technology School of Bioscience and Bioengineering South ChinaUniversity of Technology Faculty of Science Ehime University

Plasma lipoproteins are soluble complexes of proteins and lipids that transport lipids in the circulation. Historically, lipoproteins were categorized and named by density range, thus each class contains a group of molecules varying in size, charges and lipid and protein compositions. Such differences were demonstrated to contribute to their specific physiological functions and pathological roles. However, profiling of the protein content and interactions of lipoproteins has always been analytically challenging and requires methods of high sensitivity and high resolution. In our work, a method employing nondenaturing 2 de, grid gel-cutting and quantitative LC-MS/MS has been used to analyze the associated proteins in human plasma high-density lipoprotein(HDL) and low-density lipoprotein(LDL) [1,2]. A human plasma sample was separated by nondenaturing 2 de, the gel areas that were detected by immunochemical staining to contain HDL and LDL were respectively subjected for grid cut. For HDL, the 5 mm × 18 mm area was cut into 90 squares of 1 mm × 1 mm, and each of the gel piece was analyzed in standardized procedures of in-gel digestion and quantitative LC-MS/MS. Totally 154 proteins were detected and the quantity distribution of each was reconstructed as a color-density pattern(a native protein map). The map of apolipoprotein(Apo) A-I showed a wide apparent mass distribution characteristic to HDL and was compared with the maps of the other 153 proteins. Eleven proteins showed maps of wide distribution that overlapped with the map of Apo A-I, and all have been reported to be the components of HDL. Further, seven minor proteins associated with HDL were detected at the gel positions of high Apo A-I quantity. In the analysis of LDL, the 18 mm × 4.8 mm area was cut into 72 squares. A total of 253 proteins were detected and their native maps were reconstructed. Apo B-100 was shown to be the most abundant protein in the grid-cut area, concentrated at p I ca. 5.4–6.1 and appare

关键词： human lipoproteins HDL LDL nondenaturing 2de LC-MS/MS mapping

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