Four-Dimensional Orthogonal Electrophoresis System for Screening Protein Complexes and Protein−Protein Interactions Combined with Mass Spectrometry

Abstract

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 (4-DE) system, 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 candidates 20S core particle of proteasome (CP), hemoglobin (Hb, α2β2), Hb (α2δ2), peroxiredoxin-2 (PRDX2), carbonic anhydrase-1 (CAH1), and heat shock protein 60 (HSP60) were separated. CP, Hb (α2β2), PRDX2, and HSP60 with different MWs and pI's were chosen for Part II proteomic analysis. The results indicate that 4-DE 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.

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

“…The mildness of the 1st-DE and 2nd-DE basic has been confirmed in a previous study (18). The 2nd-DE acidic , activity assay of the CP was also carried out with help of basic-native-PAGE to indirectly prove the mildness of acidic-native-PAGE.…”

“…The detailed procedure was similar to that of a previous study (18) with slight modification. Briefly, 4% (w/v) thin layer gel (130 mm ϫ 100 mm ϫ 1.0 mm, acrylamide/bis-acrylamide ϭ 20:1) was prepared for IEF without denaturants such as urea and detergent, containing Ampholines at pH 3.0 -9.5 (0.5%, v/v), 3.5-10.0 (0.5%, v/v), 5.0 -7.0 (0.5%, v/v), and 6.0 -9.0 (0.5%, v/v).…”

“…The 2nd-DE: Acidic-Basic-Native-PAGE-Previous studies employed basic-native-PAGE as the second dimension electrophoresis (17,18), which provided high resolution for cytoplasmic protein separation. However, it is still incompatible with extremely basic proteins with pI above the pH of gels (Fig.…”

“…First, 0.55 L of 200 mM dithiotreitol (DTT) in 250 mM NH 4 HCO 3 was added into this extractive solution (final DTT concentration of 20 mM), and the solution was kept at 56°C for 45 min, followed by 0.62 L of 500 mM iodoacetamide (IAA) in 25 mM NH 4 HCO 3 treatment in dark at room temperature for 30 min (final IAA concentration of 50 mM). Twelve percent (w/v) separating gel of SDS-PAGE was used here as described earlier (18). Each reductive-alkylated protein extractive solution mixed with 1.6 L of 5ϫ SDS-PAGE loading buffer was loaded into the well, and electrophoresis was run at 10 mA/gel for 30 min, followed by 20 mA/gel until the bromphenol blue migrated to the bottom of the gels.…”

“…Meanwhile, as practical exploration of complexomics should also incorporate intact complexes and dissociated subunit information, so, we developed a novel approach termed the broad-spectrum four-dimensional orthogonal electrophoresis (BS4-DE) system, based on our previous fourdimensional electrophoresis (4-DE) system (18), which is mainly composed of two parts: 1st-/2nd-DE: native-thin-layer-isoelectric focusing (IEF) (native-tl-IEF)/acidic-basic-native-PAGE (nondenaturing part I), and 3rd-/4th-DE: denaturing-IEF/SDS-PAGE (denaturing part II) (Fig. 1).…”