Broad-spectrum four-dimensional orthogonal electrophoresis: a novel comprehensively feasible system for protein...

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 four-dimensional 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-thin-layer-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.