Proteomic analysis of protein expression profiles during Caenorhabditis elegans development using two-dimensional difference gel electrophoresis

Coordinated protein expression is critical for the normal execution of animal development. To obtain overall proteome profiles during animal development, a small free-living soil nematode, Caenorhabditis elegans, was used as a model and the developmental changes of protein expressions were analyzed using two-dimensional difference gel electrophoresis. Protein samples from six developmental stages were prelabeled with fluorescent cyanine dyes and separated on two-dimensional electrophoresis gels. Image-to-image analysis of protein abundances together with protein identification by peptide mass fingerprinting yielded the developmental expression profiles of 231 spots representing 165 proteins. About a quarter of the identified proteins were expressed in multiple spots with different isoelectric points, suggesting a certain proportion of proteins were variously modified. This notion was supported by the observation that about a third of the multispot proteins were stained positive for a phosphoprotein specific dye. While a fairly large number of the proteins showed little alteration in their expression profiles during development, about 40 proteins were found to be significantly either up- or down-regulated between the embryos and newly hatched L1 larvae. Down-regulated proteins included those related to the cell cycle such as MCM-7, PCN-1, and the mitotic checkpoint protein, while up-regulated proteins included structural proteins such as actins, LEV-11, DIM-1, VAB-21, metabolic enzymes such as ATP synthase, ALH-12, fluctose-1,6-bisphosphate aldolase and GPD-3, and galectins. A standard proteome map was obtained where the defects in the mutations of developmental genes and the effects of reagents on the development in C. elegans were analyzed.     

Comparison of proteomes in various human plasma preparations by two-dimensional gel electrophoresis

Serum or plasma can be utilized in a variety of studies targeted toward the discovery of disease biomarkers. In this study, the proteome profiles of plasma samples prepared using various anticoagulants (EDTA, heparin or citrate), were compared with those of serum using two-dimensional electrophoresis (2-DE). Proteins which evidenced different levels in the plasma and serum were screened and identified using ESI-Q-TOF MS/MS. The proteins which became detectable after the removal of fibrinogen from serum were identified as pigment epithelial differentiating factor (four spots), fetuin-like protein, and the hemopexin precursor. In particular, three proteins, pre-serum amyloid P component, plasma glutathione peroxidase precursor, and tetranectin, evidenced increased volume intensity only in the plasma samples prepared with EDTA.     

Optimizing protein solubility for two-dimensional gel electrophoresis analysis of human myocardium

In order to maximize the myocardial proteome observed by two-dimensional gel electrophoresis (2-DE), the effect of (1) either an ionic or different zwitterionic detergents during tissue homogenization and (2) altering the "standard" detergent for isoelectric focusing (3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS)) to either the zwitterionic detergent amidosulfobetaine-14 (ASB-14) or N-decyl-N-N'-dimethyl-3-ammonio-1-propane sulfonate (SB3-10) was investigated. Sodium dodecyl sulfate was shown to be a superior detergent for extraction of proteins during homogenization of cardiac tissue compared to the detergents ASB-14, SB3-10 or CHAPS. Additionally, both ASB-14 and SB3-10 exhibited better extraction than CHAPS for distinct regions of two-dimensional gels. In most cases, the best combination of homogenization and focusing conditions did not involve the use of the same detergent. Specifically, it was found that the ability to mix homogenization and focusing conditions can allow one to obtain an optimum balance between the resolution and number of protein spots obtained in 2-DE analysis of cardiac tissue. An excellent initial combination of buffers to utilize for the general examination of cardiac proteins was determined to be initial homogenization in a buffer containing ASB-14 followed by focusing in a buffer containing CHAPS.     

Non-covalent and covalent protein labeling in two-dimensional gel electrophoresis

Over the past decades, several sensitive post-electrophoretic stains have been developed for an identification of proteins in general, or for a specific detection of post-translational modifications such as phosphorylation, glycosylation or oxidation. Yet, for a visualization and quantification of protein differences, the differential two-dimensional gel electrophoresis, termed DIGE, has become the method of choice for a detection of differences in two sets of proteomes. The goal of this review is to evaluate the use of the most common non-covalent and covalent staining techniques in 2D electrophoresis gels, in order to obtain maximal information per electrophoresis gel and for an identification of potential biomarkers. We will also discuss the use of detergents during covalent labeling, the identification of oxidative modifications and review influence of detergents on finger prints analysis and MS/MS identification in relation to 2D electrophoresis.     

Co-activation of vastus lateralis and biceps femoris muscles in pubertal children and adults

Determining the mechanisms of co-activation around the knee joint with respect to age and sex is important in terms of our greater understanding of strength development. The purpose of this study was to examine the effects of age, sex and muscle action on moment of force and electromyographic (EMG) activity of the agonist and antagonist muscle groups during isokinetic eccentric and concentric knee extension and flexion. The study comprised nine pubertal boys [mean age 12.6 (SD 0.5) years], nine girls [12.7 (SD 0.5) years] nine adult men [23.1 (SD 2.1) years] and nine adult women [23.7 (SD 3.1) years] who performed maximal isometric eccentric and concentric efforts of knee extensors and flexors on a dynamometer at 30 degrees x s(-1). The moment of force and surface EMG activity of vastus lateralis and biceps femoris muscles were recorded. The moment of force:agonist averaged EMG (aEMG) ratios were calculated. The antagonist aEMG values were expressed as a percentage of the aEMG activity of the same muscle, at the same angle, angular velocity and muscle action when the muscle was acting as agonist. Three-way analysis of variance (ANOVA) designs indicated no significant effects of age or sex on moment:aEMG ratios. Eccentric ratios were significantly higher than the corresponding concentric ones (P < 0.05). The results also indicated no significant effect of age and sex on the aEMG of the vastus lateralis and biceps femoris muscles when acting as antagonists. The antagonist aEMG was significantly greater during concentric agonist efforts compared with the corresponding eccentric ones (P < 0.05). These findings would suggest that the moment exerted per unit of agonist EMG and the antagonist activity are similar in children compared with adults and are not sex dependent. Future comparisons between eccentric and concentric moments of force and agonist ENG should take into consideration the antagonist effects, irrespective of age or sex.     

Comparison of human and pigeon erythrocyte membrane proteins by one- and two-dimensional gel electrophoresis

Pigeon and human bands 1 and 2 (spectrin) and 5 (actin) are conserved. Band 3 anion porters have similar SDS positions, but the pigeon porter has a higher isoelectric point. Both anion porters are inhibited by similar doses of pyridoxal phosphate. Many differences are apparent in minor bands.     

Mapping of proteins in human saliva using two-dimensional gel electrophoresis and peptide mass fingerprinting

Human saliva contains a large number of proteins that can be used for diagnosis and are of great potential in clinical and epidemiological research. The aim of this work was to map the proteins in saliva by two-dimensional gel electrophoresis (2-DE), and to identify abundant proteins by peptide mass fingerprinting using trypsin cleavage and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry analysis. One hundred proteins were identified representing 20 different identities according to accession numbers. Abundant proteins expressed in different forms were: alpha-amylase, immunoglobulin A, prolactin-inducible protein, zinc-alpha(2)-glycoprotein and cystatins (S, SA, D and SN). Other proteins found were interleukin-1 receptor antagonist, von Ebner's gland protein (lipocalin-1) and calgranulin A and B (S100A8 and A9). Furthermore, apolipoprotein A-I, beta(2)-microglobulin, glutathione S-transferase P and fatty acid-binding protein were also identified. Our results show that human saliva contains a large number of proteins that are involved in inflammatory and immune responses. The 2-DE protein map constructed opens the possibility to investigate protein changes associated with disease processes.     

Quantitative analysis of mitochondrial protein expression in methylmalonic acidemia by two-dimensional difference gel electrophoresis

Isolated methylmalonic acidemia (MMA) is a rare metabolic disease due to the deficient activity of L-methylmalonyl-CoA mutase (MCM). This mitochondrial enzyme converts L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin (Adocbl) as cofactor. Isolated MMA is subdivided into five forms: mut MMA associated with MCM deficiency, three different defects related to mitochondrial Adocbl formation (cblA, cblB, and cblH), and cblD variant 2. We performed proteomic analysis on mitochondria from an individual with cblH/cblD disorder using 2-D DIGE to identify differentially expressed proteins in this disease. Comparative analysis of control/patient mitochondrial proteome allowed us to identify differential expression of 10 proteins. The most notable groups included proteins involved in apoptosis (cytochrome c), oxidative stress (manganese superoxide dismutase) and cell metabolism (succinyl-CoA ligase (GDP forming) and mitochondrial glycerophosphate dehydrogenase). Immunoblot analysis further validated 2-D DIGE results of two of these proteins in multiple MMA patients, suggesting that the differences in expression are a general effect in this disorder. It is feasible that the differential proteins identified in this study have a biological significance and might be related to the pathophysiology of MMA.     

Determining a significant change in protein expression with DeCyder during a pair-wise comparison using two-dimensional difference gel electrophoresis

Two-dimensional difference gel electrophoresis (DIGE) is a tool for measuring changes in protein expression between samples involving pre-electrophoretic labeling with cyanine dyes. Here we assess a common method to analyze DIGE data using the DeCyder software system. Experimental error was studied by a series of same sample comparisons. Aliquots of sample were labeled with N-hydroxyl succinimidyl ester-derivatives of Cy2, Cy3, and Cy5 dyes and run together on one gel. This allowed assessment of how experimental error influenced differential expression analysis. Bias in the log volume ratios was observed, which could be explained by differences in dye background. Further complications are caused by significant gel-to-gel variation in the spot volume ratio distributions. Using DeCyder alone results in an inability to define ratio thresholds for 90 or 95% confidence. An alternative normalization method was thus applied which resulted in improved data distribution and allowed greater sensitivity in analysis. When combined with a standardizing function, this allowed gel-independent thresholds for 90% confidence. The new approach, detailed here, represents a method to greatly improve the success of DIGE data analysis.     

Markers for human placental trophoblasts in two-dimensional gel electrophoresis

Summary Primary cultures of trophoblasts established from human term placentae showed high viability and reproducibility. Two-dimensional gel patterns obtained by metabolically labeling the trophoblasts with [³⁵S]-methionine demonstrated that their pattern of gene expression was stable during the 6-d period investigated. Gel analysis demonstrated the keratins 7, 8, 14, 17, 18, and 19. Analysis of the gel pattern confirmed the presence of a small proportion of contaminating fibroblasts and lymphocytes. The gel patterns were compared with that of skin fibroblasts, peripheral lymphocytes, and epithelial cells to identify a group of proteins that are enriched in the trophoblasts and thus may be used as marker for these cells. This work was supported by the Danish Cancer Society, the Lundbeck Foundation, the Danish Medical Research Council, “Pedersholmlegatet,” and by “Anna og Jakob Jakobsens Legat.”     

Comparison of protein constituents between atria and ventricles from various vertebrates by two-dimensional gel electrophoresis

1. Protein constituents of cardiac muscles of 23 species were examined by two-dimensional gel electrophoresis in order to find the difference in protein components between atria and ventricles. 2. Protein compositions of atria were similar to those of ventricles, however, differences were found in myosin and some other proteins in most species. 3. A major protein with molecular weights of 12,000-15,000 daltons was distributed only in atria from mammals. 4. The atrioventricular difference suggested that the ventricular tissue was more specialized in mammals than in birds, as compared with the atrial tissue.     

Fibre conduction velocity and fibre composition in human vastus lateralis

The relationship between muscle fibre composition and fibre conduction velocity was investigated in 19 male track athletes, 12 sprinters and 7 distance runners, aged 20-24 years, using needle biopsy samples from vastus lateralis. Cross sectional areas of the fast twitch (FT) and slow twitch (ST) fibres were determined by histochemical analysis. The percentage of FT fibre areas ranged from 22.6 to 93.6%. Sprinters had a higher percentage of FT fibres than distance runners. Muscle fibre conduction velocity was measured with a surface electrode array placed along the muscle fibres, and calculated from the time delay between 2 myoelectric signals recorded during a maximal voluntary contraction. The conduction velocity ranged from 4.13 to 5.20 m.s-1. A linear correlation between conduction velocity and the relative area of FT fibres was statistically significant (r = 0.84, p less than 0.01). This correlation indicates that muscle fibre composition can be estimated from muscle fibre conduction velocity measured noninvasively with surface electrodes.     

Targeting of Bacillus anthracis interaction factors for human macrophages using two-dimensional gel electrophoresis

Bacillus anthracis, a gram-positive, endospore-forming, aerobic rod-shaped bacterium, interacts with macrophages at various stages of the disease. Spore germination and the outgrowth of vegetative bacilli are crucial steps enabling the bacteria to proliferate actively and to synthesize the virulence factors leading to a massive septicemia. In this study, we performed a proteomic analysis and MALDI-TOF/MS were carried out to identify proteins using human macrophages infected with the spores of B. anthracis live-Sterne or inactivated-Sterne. We identified 21 proteins which are related to the infection of B. anthracis spores on human macrophages at the early stage events. These proteins function in processes such as cytoskeleton regulation, apoptosis, cell division, and protein degradation. Proteins such as PAK 2 revealed a relationship to apoptosis in human macrophages. These proteins play an important role in the macrophage survival and death on human macrophages with infected B. anthracis spores.     

Very-high-resolution two-dimensional gel electrophoresis of proteins using giant gels

An improved high-resolution two-dimensional gel system for separating complex protein mixtures is described that allows a threefold increase in the number of proteins detected. Like the original O'Farrell system, proteins are separated in the first dimension by isoelectric point and in the second dimension by size. The improved resolution results primarily from a 2.5-fold increase in the size of both dimensions. Although best resolution is obtained by application of <100 μg of protein containing >5 × 10⁶ cpm, as much as 150 μg of protein may be applied without appreciable loss of resolution. Useful separations may be made with up to 1.5 mg. By doubling the thickness of both dimensions, as much as 3 mg of protein can be separated into 300–400 separate peaks.     

Monitoring intracellular protein profiles with two-dimensional gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is a method of separating complex protein mixtures, such as whole cell extracts, on the basis of protein isoelectric point and molecular weight. In bioprocess engineering, conventional 2D PAGE has tremendous potential to yield detailed information on the intracellular effect of various process conditions. It has been used in our work to examine global intracellular changes occurring in a typical cycloheximide fermentation and to look at the feedback regulatory behavior of cycloheximide biosynthesis. Application of the technique for bioprocess monitoring will require that the time necessary for preparation of a 2D electropherogram be substantially shortened. This may be accomplished by performing the separation on a miniature scale or eventually by use of capillary electrophoresis for one or more of the separations. Advantages and disadvantages of these two approaches are discussed.     

Analysis of tubulin isoforms by two-dimensional gel electrophoresis using SDS-polyacrylamide gel electrophoresis in the first dimension.

A two-dimensional electrophoretic system using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the first dimension and isoelectric focusing (IEF) in the second dimension was devised. In spite of its simplicity, this method could show a markedly high resolution for tubulin isoforms and moreover could classify them into alpha- or beta-tubulin as a two-dimensional profile. With this method, seven alpha- and four beta-tubulin isoforms could be detected within axoneme from Tetrahymena cilia. Moreover this method could also resolve tubulin isoforms from the rabbit brain. These results indicate that the present two-dimensional gel electrophoresis is a useful tool for the electrophoretic analysis of tubulin isoforms from various sources.