| Protein molecules interact with each other to exert their biological functions, therefore, proteomic techniques should be able to analyze not only the structures and quantities of the constituent proteins, but their interactions, such as covalent bonding of subunits, noncovalently bound multimers, protein complexes with multiple enzyme functions, etc. In this work, a new native proteomic strategy combining nondenaturing micro two-dimensional gel electrophoresis(2DE), grid gel-cutting and quantitative nano ultra-performance liquid chromatography-tandem mass spectrometry(nano-UPLC-MS/MS) has been developed employing soluble proteins of human bronchial smooth muscle cells(HBSMC) as a model proteome system, aiming visualization of distribution patterns of cellular proteins on a nondenaturing 2D gel and large-scale prediction of protein-protein interactions.The analysis comprised of three procedures that were performed sequentially, namely, 1) extraction of HBSMC soluble proteins followed with the separation by nondenaturing micro 2DE; 2) grid-cutting of the major area of the gel(30 mm × 40 mm) into nine hundreds and seventy two 1.1 mm × 1.1 mm(1.0 mm thick) squares; 3) analysis of the proteins in each of the 972 square gel pieces by procedures of in- gel digestion, peptide extraction, supplementation of quantitative internal standard and quantitative nano-UPLC-MS/MS(label- free). The results showed that each o f the gel piece provided the assignment of 2 to 280 proteins(average ca. 80), together with the information of their quantities. 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 × 10-5% of the total protein quantity in the grid area. Each protein map was characterized by several features, including the position of qua ntity peak square, number of detected squares, and degree of concentration(focused or dispersed). 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 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, which would facilitate further analysis of the functions of the proteins.With the logic that proteins comprising a protein complex would be separated together under nondenaturing conditions, we developed a method to evaluate the degree of similarity between the protein maps for the analysis of interactions. The following procedure was employed using laboratory-made Excel Visual Basic(VB) macros;(i) maps which have 3 or more squares with protein q uantity data were selected(2328 native maps out of the 4323),(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 which showed overlap factors above 0.65 from both protein sides were selected(431 protein pairs). Each protein pair was searched in a database(Uni Prot KB) 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 cel s.In conclusion, we here developed a novel native proteomic strategy, combining nondenaturing micro 2DE, grid gel-cutting and quantitative LC-MS/MS, and successfully applied it to a eukaryotic cellular proteome(HBSMC soluble proteins). Totally 4323 proteins were assigned and a native map of each was reconstructed from the quantity data using laboratory- made Excel VB macros. An algorithm to evaluate the degree of similarity between protein maps with 3 or mo re detected squares(2328 maps) was developed introducing the concept of “overlap factor” and a matrix of overlap factors(2328 × 2328) was prepared to select protein map pairs with high similarity. O ut of the selected 431 pairs with the overlap factors from both sides over 0.65, 301 protein pairs were found to be documented in a database Uni Prot KB to form protein complexes to comprise 35 protein complexes and one protein complex was newly proposed in human, demonstrating that the similarity comparison method enabled simultaneous analysis of multiple cellular protein complexes on nondenaturing 2D gels. We expect that using mass spectrometer with higher sensitivity in protein identification would enable to detect the low-abundant proteins in larger numbers of squares and expand the applicability of the overlap search method to all 4323 proteins or more proteins.It is noteworthy that we found there were nearly 30% of the proteins detected in the soluble fraction being documented as membrane proteins in datab ase, suggesting that with improved solubilization of membrane proteins this native proteomic strategy has the potential to be used for wholesome proteomic analysis. |
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