title: Analysis of low-density lipoprotein-associated proteins using the method of digitized native protein mapping.
abstract: The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS (in data-independent acquisition mode, or MS(E) ), was improved by using a new MS/MS mode, ion mobility separation enhanced-MS(E) (HDMS(E) ), and applied to analyze the area of human plasma low-density lipoprotein (LDL). An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid-cut into 72 square gel pieces and subjected to quantitative LC-MS/MS. Compared with MS(E) , HDMS(E) showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. A total of 253 proteins were assigned with LC-HDMS(E) and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B-100 was the most abundant protein in the grid-cut area, concentrated at pI ca. 5.4-6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39-42. An Excel macro was prepared to search protein maps which showed protein quantity peaks localized within this concentrated region of Apo B-100. Twenty-two proteins out of the 252 matched this criterion, in which 19 proteins have been reported to be associated with LDL. This method only requires several microliters of a plasma sample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL.
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, P. R. China.|Pre-Incubator for Innovative Drugs & Medicine, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, P. R. China.
authors.forename: Ya
authors.initials: Y
authors.lastname: Jin
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, P. R. China.|Pre-Incubator for Innovative Drugs & Medicine, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, P. R. China.
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, P. R. China.|Pre-Incubator for Innovative Drugs & Medicine, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, P. R. China.
authors.forename: Ahui
authors.initials: A
authors.lastname: Wang
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, P. R. China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, P. R. China.|Pre-Incubator for Innovative Drugs & Medicine, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, P. R. China.
authors.forename: Shumin
authors.initials: S
authors.lastname: Chen
authors.affiliation: Faculty of Science, Ehime University, Matsuyama, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Manabe
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, P. R. China.|Pre-Incubator for Innovative Drugs & Medicine, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, P. R. China.|Key Laboratory of Industrial Biotechnology of Guangdong Higher Education Institutes, South China University of Technology, Guangzhou, P. R. China.
authors.forename: Wen
authors.initials: W
authors.lastname: Tan
mesh_terms: D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D008077:Lipoproteins, LDL / Q000032:analysis*; D053719:Tandem Mass Spectrometry
chemical_list: D008077:Lipoproteins, LDL
keywords: Digitized native protein mapping; Human plasma low-density lipoprotein; Nondenaturing micro 2DE; Protein-protein interactions; Quantitative LC-HDMSE