Broad-spectrum Four-dimensional Orthogonal Electrophoresis: A Novel Comprehensively Feasible System for Protein Complexomics Investigation

Abstract

The major challenge of "protein complexomics" is to separate intact protein complexes or interactional proteins without dissociation or denaturation from complex biological samples and to characterize structural subunits of protein complexes. To address these issues, we developed a novel approach termed "broad-spectrum fourdimensional orthogonal electrophoresis (BS4-DE) system," which is composed of a nondenaturing part I and denaturing part II. Here we developed a mild acidic-native-PAGE to constitute part I, together with native-thinlayer-IEF and basic-native-PAGE, widening the range of BS4-DE system application for extremely basic proteins with the range of pI from about 8 to 11 (there are obviously 1000 kinds of proteins in this interval), and also speculated on the mechanism of separating. We first proposed ammonium hydroxide-ultrasonic protein extractive strategy as a seamless connection between part I and part II, and also speculated on the extractive mechanism. More than 4000 protein complexes could be theoretically solved by this system. Using this approach, we focus on blood rich in protein complexes which make it challenging to sera/ plasma proteome study. Our results indicated that the BS4-DE system could be applied to blood protein complexomics investigation, providing a comprehensively feasible approach for disease proteomics. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.012450, 786 -799, 2012.In the postgenomic era, protein complexomics highlights its prominent role in proteomics. Characterization of biologically important protein complexes can provide an integrative view of the protein-protein interactive networks that reveal protein function and biological behavior. Despite enormous progresses in two-step affinity purification (1), comprehensive two-hybrid (2, 3), high-throughput yeast two-hybrid (4, 5), co-immunoprecipitation (co-IP) (6), and high-throughput coaffinity purification followed by mass spectrometry (MS) (7) for separating and characterizing protein complexes, the multistep process is liable to lead to the dissociation and even denaturation of protein complexes. Convenient approaches that could be used for direct study of protein complexes are, as yet, lacking.Various forms of mild electrophoresis have for some time been the best tools for directly analyzing protein complexes, such as the charge shift techniques: blue native electrophoresis (BNE) 1 (8) and high resolution clear native electrophoresis (hrCNE) (9), offering clear advantages for hydrophobic protein complex analyses, especially for membrane protein complexes. However, BNE is not suitable for detergent-labile assemblies' analyses and in-gel catalytic activity assays because of the use of anionic detergents and Coomassie brilliant blue (CBB) dye. Also, hrCNE has been proven successful in some but not in all aspects (9). Clear native electrophoresis (CNE) (10), in contrast, has only recently been recognized as a valuable and milder technique to isolate and functionally investigate multiprotein complexes (11,12). Ho...

Elevated Serum Levels of Circulating Immunoinflammation-Related Protein Complexes Are Associated with Cancer

“…The major components of these protein complexes are listed in Table . Interestingly, there are few IIRPCs in pattern c samples, which may be due to different haptoglobin phenotypes of the serum …”

“…Our findings are biologically reasonable. Haptoglobin is one of the acute phase reactants and inflammatory markers, and it has also been found to be a cancer-associated protein . Complement proteins contribute to both innate and adaptive immunity and play an essential role in the development of inflammation .…”

“…Currently, the measurement of these protein complexes is mainly performed by immunoassays, which have the disadvantage of limiting the exploration of biomarkers to known protein complexes. To overcome the above limitations, native-polyacrylamide gel electrophoresis (native-PAGE), − combined with mass spectrometry-based proteomic technologies, has been used to isolate and identify serum protein complexes and cancer-associated protein complexes …”

“…To overcome the above limitations, nativepolyacrylamide gel electrophoresis (native-PAGE), 8−10 com-bined with mass spectrometry-based proteomic technologies, has been used to isolate and identify serum protein complexes and cancer-associated protein complexes. 11 Both chronic inflammatory processes and humoral immune responses are associated with many human cancers. 12−14 Only a few studies to date have tried to correlate humoral immune responses with cancer on a large number of samples.…”

A Caffeic Acid Matrix Improves In Situ Detection and Imaging of Proteins with High Molecular Weight Close to 200,000 Da in Tissues by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

“…Protein Extraction and SDS-PAGE Analysis. The detailed processes of protein extraction from three selected biological tissues (a Capparis masaikai seed, a rat brain, and a germinating soybean seed) and SDS-PAGE separation 45 are shown in the Supporting Information Experimental Section and Tables S1 and S2.…”