Protein Complexes And Protein Interaction Studies Of A New Strategy

Most current approaches for purification and identification of protein complexes adopt affinity purifications combined with mass spectrometry, such as co-immunoprecipitation and tandem affinity purification. Herein, we propose a new approach, termed as the four-dimensional orthogonal electrophoresis system (4-DES), to find and analyze the cytoplasmic protein complexes.4-DE system is composed of two parts:nondenaturing part (Part Ⅰ) and denaturing part (Part Ⅱ). Through Part Ⅰ and decision procedure separations, six protein complex candidates20S core particle of proteasome (CP), hemoglobin (Hb, α2β2), Hb (α2δ2), peroxiredoxin-2(PRDX2), carbonic anhydrase-1(CAH1), and heat shock protein60(HSP60) were separated. CP, Hb (α2β2), PRDX2, and HSP60with different MW's and pI's were chosen for Part Ⅱ proteomic analysis. The results indicate that4-DES is not only suitable for studying protein complexes and protein-protein interactions as well as structural proteomics from complex biological samples, but can also be easy to separate and concentrate intact protein complexes from dilute complex samples. The major challenges of 'protein complexomics'are 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 as "broad-spectrum four-dimensional orthogonal electrophoresis system (BS4-DES)", which is composed of nondenaturing part Ⅰ and denaturing part Ⅱ. Here we developed a mild acidic-native-PAGE to constitute part Ⅰtogether with native-thin-layer-IEF and basic-native-PAGE, widening the range of BS4-DES application for extremely basic proteins with the range of pI from about8to11, there are obviously1,000kinds of proteins in this interval, and also speculated on the separating mechanism. 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 than4,000protein 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-DES could be applied to blood protein complexomics investigation, providing a comprehensively feasible approach for disease proteomics.