Detection of protein‐protein interactions and a group of immunoglobulin G‐associated minor proteins in human plasma by nondenaturing and denaturing two‐dimensional gel electrophoresis

Abstract

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.

Cleavage of fibrinogen alpha chains during isoelectric focusing of human plasma under non-denaturing conditions analyzed by micro two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry

“…1B, which were obvious only after silver staining, would not be suited for the analysis. Previously, we have reported that the minor proteins are likely to form high-molecular-mass proteins under non-denaturing conditions and they are present in 0-4% PEG fractions or in 0-35% AS fractions 1) . Then we employed AS fractionation to enrich them and the 0-35% AS fraction (described in Section 2.2, ca.…”

“…When IEF was run with electrode solutions kept around 30°C and the 2-DE gel was stained with CBB, human plasma proteins were separated as shown in Figure 1A. The minor spots which had been detected at pI 5.5-7.5 and mass 8-45 kDa 1) are not obvious in the 2-DE pattern, but when the gel was further stained with silver ( Fig. 1B), more than 50 spots were detected in the corresponding region.…”

“…Previously, we have reported a group of minor proteins in human plasma which were not detected when a plasma sample was subjected to non-denaturing isoelectric focusing (IEF) followed by non-denaturing pore-gradient gel electrophoresis (Type-I 2-DE) 2) , but detected when it was subjected to non-denaturing IEF followed by SDS gel electrophoresis (Type-II 2-DE) 2) and suggested that they might participate in blood coagulation 1) . Since the minor proteins could be enriched in 0-35% saturated ammonium sulfate fraction of human plasma, the fraction was subjected to Type-II 2-DE employing micro 2-DE system.…”

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

“…However, lower resolution and narrower separation of basic proteins or complexes are major limitations of CNE compared with BNE or hrCNE (13). Nondenaturing micro-two dimensional electrophoresis (micro 2-DE) was developed later to overcome the lower resolution of CNE (14). Although it has been used widely in plasma and cytoplasm protein complex investigations (15)(16)(17), it is still not suitable for basic protein analysis with a pI above the pH of the gel.…”

Alkaline cleavage of covalent bonds in chicken insulin and bovine ?-lactalbumin analyzed by matrix-assisted laser desorption/ionization- mass spectrometry

“…The samples after different duration of alkaline treatment, but containing the same quantity of a protein (1.0 mg), were subjected to SDS-PAGE employing micro slab gels (38 mm638 mm61 mm, 8.4%-17.85% T and 5% C polyacrylamide gradient gel), then the gels were stained with CBB R-250 and destained [13]. Quantitation of the stained protein bands on the gel was performed using a scanner and an image analysis software (Phoretics 2-D Full; Nonlinear Dynamics, Newcastle, UK) [14]. …”