Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC‐MS/MS. 2. Similarity search between protein maps for the analysis of protein complexes

Abstract

Human bronchial smooth muscle cell soluble proteins were analyzed by a combined method of nondenaturing micro 2DE, grid gel‐cutting, and quantitative LC‐MS/MS and a native protein map was prepared for each of the identified 4323 proteins [1]. A method to evaluate the degree of similarity between the protein maps was developed since we expected the proteins comprising a protein complex would be separated together under nondenaturing conditions. The following procedure was employed using Excel macros; (i) maps that have three or more squares with protein quantity data were selected (2328 maps), (ii) within each map, the quantity values of the squares were normalized setting the highest value to be 1.0, (iii) in comparing a map with another map, the smaller normalized quantity in two corresponding squares was taken and summed throughout the map to give an “overlap score,” (iv) each map was compared against all the 2328 maps and the largest overlap score, obtained when a map was compared with itself, was set to be 1.0 thus providing 2328 “overlap factors,” (v) step (iv) was repeated for all maps providing 2328 × 2328 matrix of overlap factors. From the matrix, protein pairs that showed overlap factors above 0.65 from both protein sides were selected (431 protein pairs). Each protein pair was searched in a database (UniProtKB) on complex formation and 301 protein pairs, which comprise 35 protein complexes, were found to be documented. These results demonstrated that native protein maps and their similarity search would enable simultaneous analysis of multiple protein complexes in cells.

Analysis of low‐density lipoprotein‐associated proteins using the method of digitized native protein mapping

“…However, we designated the term “native protein mapping” to express the process to reconstruct a book of protein distribution maps obtained by the method described here. The book contains hundreds of pages of individual protein maps in the case of an area of a nondenaturing 2D gel of human plasma proteins and thousands of pages of individual protein maps in the case of an area of a nondenaturing 2D gel of cellular proteins . In the analysis of the area which contained plasma LDL, we obtained 164 maps using MS E MS/MS mode and 253 maps using HDMS E MS/MS mode.…”

A comparative analysis of human plasma and serum proteins by combining native PAGE, whole‐gel slicing and quantitative LC‐MS/MS: Utilizing native MS‐electropherograms in proteomic analysis for discovering structure and interaction‐correlated differences

“…The apparent masses of the proteins detected in each of the 35 squares, numbered 1 to 35 from the gel top, were estimated from a standard curve of log (apparent mass) versus (migration distance), as we have reported on the analysis of human plasma proteins using nondenaturing 2DE . It must be noted that proteins with p I values closer to the running buffer (pH 8.3) would have smaller mobility than the estimated apparent masses of the corresponding gel squares .…”

“…Detection of gel‐separated proteins is generally done through global detection by staining with a dye such as CBB, silver or a fluorescent reagent, or detection on specific proteins by enzymatic activity staining or immunochemical staining. Densitometric analysis based on optical density measurement of dye‐stained protein bands is usually followed for quantitation, but despite the long history of usage, it has obvious limits; for global staining using a dye, on one side, detection of low‐abundant proteins is often limited by the sensitivity of the stain method so that an area not obviously stained is often overlooked, while on the other hand, a quantity calculated from an optical density is often the sum of many proteins residing at the same gel position ; as for detection of specific proteins, the specificity and quantitation performance of the immunochemical or activity staining method is often insufficient .…”

“…Abundance‐mass profiles of 774 rat brain mitochondrial proteins were obtained and further used to study the assembly of respiratory chain complexes . We have been working on native protein mapping and interaction analysis by combining nondenaturing micro 2DE, gel grid‐cutting and quantitative LC‐MS/MS . Quantity distributions of more than 4000 proteins on a nondenaturing micro gel, which was grid‐cut into 972 squares with dimensions of 1.1 mm × 1.1 mm, were reconstructed for human bronchia smooth muscle cells.…”

“…Quantity distributions of more than 4000 proteins on a nondenaturing micro gel, which was grid‐cut into 972 squares with dimensions of 1.1 mm × 1.1 mm, were reconstructed for human bronchia smooth muscle cells. Comparison analysis of the distributions, referred as native protein maps, provided 431 protein pairs with high similarity, among which 301 pairs comprising 35 protein complexes were found to be documented in UniProtKB . By applying the method to a certain gel area, we analyzed the protein distributions in the regions of human plasma high‐density lipoprotein (HDL) and low‐density lipoprotein (LDL) and investigated protein interactions in the two protein complexes .…”