We analyzed the differential expression pattern of intracellular proteins in tumor necrosis factor-α (TNF-α)-sensitive KDH-8/YK and TNF-α-resistant cKDH-8/11 rat hepatoma cell lines by means of two-dimensional electrophoresis (2-DE). The 2-DE protein patterns of KDH-8/YK cells demonstrated an increase in the protein spot of relative molecular mass (Mr) 36,000 and isoelectric point (pI) 4.9, which was determined by mass spectrometry (MS). Peptide mass fingerprinting (PMF) suggested that aldose reductase (AR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and tropomyosin were the candidate components of the spot. By immunoblot using specific antibodies, the spot was identified to be AR with a pI that was lower than the theoretical value.

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We investigated whether the electrophoretic patterns and clinical information derived from the five principal fractions of the Beckman Paragon capillary zone electrophoresis (CZE) 2000 system are comparable to the results obtained by the cellulose acetate electrophoresis (CAE: Olympus AES 320) system. Concordance studies between the results obtained by CAE and CZE were performed on sera from pregnant women and from patients with nephrotic syndrome, acute and chronic inflammatory diseases, cirrhosis, monoclonal and polyclonal gammopathy, and bisalbuminemia. In all subjects except for the pregnant women, the results of CAE and CZE agreed 100% in terms of percent composition for albumin, and α₁- and α₂-globulins. Concordance for γ-globulin was 92-100% in terms of both percent composition and absolute concentration. Three discordant cases according to the results for β-globulin by CAE and CZE were found in patients with monoclonal gammopathy, who also showed discordant results for γ-globulin. An M-protein spike observed in either the β-globulin or γ-globulin fraction was 100% detected by both CAE and CZE analyses. In another six discordant cases for the β-globulin fraction observed in patients with cirrhosis and polyclonal gammopathy, part of the tail of the β-globulin fraction tended to be measured as γ-globulin by CAE. In three of the four bisalbuminemia cases, the double band of albumin observed by CZE was not detected by CAE. The results show that the electrophoretic patterns and clinical information derived from the five principal fractions of the Beckman Paragon CZE 2000 system are comparable to the results obtained by classical CAE.

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The utility of an automated 96-capillary electrophoresis instrument equipped with a laser-induced fluorescence detector was evaluated in the analysis of affinophoresis using concanavalin A and trypsin as models. Affinophores for concanavalin A and trypsin were prepared by coupling the affinity ligands, p-aminophenylmannoside and p-aminobenzamidine to soluble succinylpolylysine, respectively at a density of about 1/5 of the carboxyl groups. A dual buffer system, 60 mM borate-Na (pH 9.35) as an electrophoresis buffer and 60 mM 3-morpholinopropanesulfonic acid-Na (pH 7.35) containing 0.1% Tween 20 as a sample buffer, was employed to allow the interactions to occur at a physiological pH and also to maintain a high level of electroosmosis and reproducibility in the bare silica capillaries. The electrophoresis of the fluorophore-labeled proteins was carried out in the presence of affinophores, and changes in the mobility of the labeled proteins were analyzed in terms of the mobility moment, which represents the average mobility of the proteins, thus permitting the dissociation constants for the protein-affinophore interactions to be determined. The affinophoresis system was then used to evaluate the extent of binding of low molecular mass compounds based on the inhibition of the affinophoresis. In combination with the mobility moment analysis and the dual buffer system, the multi-capillary electrophoresis instruments proved to be useful for the analysis of biomolecular interactions by electrophoresis.

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Proteomic analysis was performed on the midgut of fifth-instar day-3 female silkworm (Bombyx mori, strain p50) larvae. Though silkworm genome analysis has not yet been completed, the Drosophila genome and silkworm expression sequence tag (EST) data were applied to the analysis of the midgut proteins in the database (DB) for identification. The spots, which were excised manually from two-dimensional electrophoresis (2-DE) gels, were treated with 4-vinylpyridine for alkylation. Each spot was analyzed by capillary high-performance liquid chromatography coupled with ion-trap mass spectrometry (MS) after proteolysis using a trypsin. Nearly 60% of the proteins analyzed by MS were identified. These included many kinds of cytoskeleton proteins, ATPase, and chaperonins.

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