Two-dimensional electrophoresis of membrane proteins

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes.     

Two-dimensional electrophoresis of membrane proteins: separation of myelin proteins

A method for two-dimensional electrophoretic separation of myelin proteins is presented. The first dimension consists of isoelectric focusing of lyophilized and delipidated membrane proteins, solubilized in a mixture of the nonionic detergent Triton X-100, the zwitterionic detergent CHAPS, 9 M urea and carrier ampholytes, and incorporated into a slab gel before separation. Subsequent discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed by moulding the isoelectric focusing slab gel with its supporting glass plate into the stacking gel. This method proved to give highly reproducible results since mechanical forces and thus the risk of stretching, folding or rupture of the isoelectric focusing slab gel is minimized. Furthermore, by immunoblotting, the positions of myelin-associated glycoprotein and 2',3'-cyclic nucleotide 3'-phosphodiesterase were established with specific antibodies.     

Two-dimensional electrophoresis of heart muscle proteins in human cardiomyopathies

Human heart muscle proteins have been analyzed by two-dimensional electrophoresis. Twenty five autopsy heart muscle samples obtained from individuals who had died in accidents and who had no signs of cardiovascular pathology have been compared with biopsy and autopsy myocardium samples of patients with: dilated cardiomyopathy (5 cases), hypertrophical cardiomyopathy (2 cases) and myocarditis (2 cases). In dilated cardiomyopathy in 3 out of 5 cases an additional protein spot was found in the myocardial myosin light chain 1 area.     

Two-dimensional electrophoresis of dog bronchoalveolar lavage fluid proteins

Proteins of dog bronchoalveolar lavage fluid, obtained by washing the epithelial lining layer of lungs with phosphate-buffered saline, were separated by two-dimensional electrophoresis. Due to the low protein and high salt content of the bronchoalveolar lavage fluid, samples had to be concentrated and desalted. Following electrophoresis the protein spots were visualized by silver staining. Comparing the two-dimensional protein patterns of bronchoalveolar lavage fluid with that from serum, several lung-specific proteins were detected. The most prominent protein, most probably a surfactant-associated protein, showed isoforms with isoelectric points in the range of pH 4.2-4.8, and a molecular mass of 32 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction with dithiothreitol.     

Two-dimensional electrophoresis of human lymphocyte proteins: two-dimensional polymorphisms and paternity testing

Genetic polymorphisms of seven human lymphocyte proteins, analyzed by two-dimensional electrophoresis, were evaluated in respect to their suitability for paternity testing. Current data of an enlarged family and population study for five proteins (p23, p30, p40, p60, p66), already described for a smaller population sample of Southern Germany, are presented together with evidence for a new polymorphic protein (p42), recently observed in our survey. These six proteins occurred in isoelectric focusing as two different variants, acidic (a) and basic (b). The genetic basis of the protein variations was ascertained (i) by the presence of homozygous and heterozygous phenotypes, (ii) by the Mendelian mode of transmission of the variants as allelic gene products within 17 families and (iii) by the demonstration of a gene-dosage dependence comparing the spot intensities in homozygous and heterozygous phenotypes. For quantitative data, laser densitometric scanning of the protein spots followed by computer-assisted quantitative evaluation of the spot intensities was performed. The allele frequencies of the polymorphic protein were calculated from the phenotype distributions within a sample of 56 unrelated individuals from Southern Germany. Gene frequencies of the common alleles ranged between 0.991 and 0.518. To discuss the suitability of the two-dimensional polymorphisms for paternity testing the theoretical exclusion probabilities were assessed for seven polymorphic proteins observed in our population sample, the six polymorphisms with two alleles described here and a further polymorphism (p75) with six alleles. For five proteins (p23, p40, p42, p66 and p75) we found sufficiently high values for the theoretical exclusion probabilities, ranging from 10% to 34%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-dimensional electrophoresis of human serum proteins following acute myocardial infarction

Serum proteins associated with acute myocardial infarction (AMI) have been monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high resolution two-dimensional electrophoresis (2-DE) under nonreducing conditions. Proteins a, b, c (Mr 13,000; pI6.2, 6.7 and 7.5, respectively) and e(Mr27,000; pI5.2) appear simultaneously approximately 30 h after infarction, reach maximum intensity after 48 h and progressively decline thereafter. Protein d (Mr15,000; pI7-8.5; identified as hemoglobin) sometimes appears within 18 h of infarction. Proteins a-c are not detected in the 2-DE patterns of healthy myocardium, infarcted myocardium, pectoral muscle or tongue, but e is present in all and tentatively identified as myosin light chain. Other myocardial proteins which are either reduced in amount following infarction or more specifically associated with myocardium than pectoral muscle are not detected in the serum of AMI patients. Analysis of unconcentrated urine by SDS-PAGE and silver staining does not reveal proteins specific to AMI.     

Two-dimensional electrophoresis of membrane proteins: A current challenge for immobilized pH gradients

Membrane proteins were separated by high resolution two-dimensional (2-D) electrophoresis. On isoelectric focusing (IEF) with immobilized pH gradients severe protein losses in the resulting 2-D map were observed when compared with carrier ampholyte-based IEF. This has been noticed for two different biological systems, namely the chloroplast envelope of spinach and the endocytic vesicles from Dictyostelium discoideum. The possible mechanisms of these losses on immobilized pH gradients are discussed.     

Two-dimensional lectin affinity electrophoresis of alpha-fetoprotein: characterization of erythroagglutinating phytohemagglutinin-dependent microheterogeneity forms

By means of two-dimensional lectin affinity electrophoresis of human alpha-fetoprotein (AFP) from different sources, AFP bands separated with erythroagglutinating phytohemagglutinin (E-PHA) were further characterized with other lectins of known oligosaccharide specificities. The results with a cord serum AFP revealed that not only AFP-P2 (E-PHA-nonreactive) but also AFP-P4 and P5 (E-PHA-reactive) had affinities for Concanavalin A (Con A) and Allomyrina dichotoma lectin (allo A), indicating that the cord serum AFP has nonbisected biantennary complex-type oligosaccharides with the terminal galactose on Man alpha 1----6 residue sialylated at the C-6, but not C-3, position. On the other hand, the results with a hepatoblastoma (HUH-6 C1-5 cell line) AFP showed that not only AFP-P5 but also AFP-P1 (E-PHA-nonreactive) and P3 (E-PHA-less reactive) had Con A-nonreactive AFP and that AFP-P1 had AFP-A1 (allo A-nonreactive) and AFP-A2 (allo A-less reactive), and AFP-P3 and P4 had AFP-A1s (allo A-nonreactive), as main components, in addition to the spots of cord serum AFP. Most of the E-PHA-dependent bands of AFP were further subdivided with Lens culinaris agglutinin (LCA-A) into LCA-A-reactive, weakly reactive and nonreactive spots. Similar results were obtained with AFP preparations from hepatocellular carcinomas and other malignancies, indicating that the bisected bi-(or tri- and tetra-) antennary sugar chains with the exposed terminal galactose of the Man alpha 1----6 arm as well as those with the C-3 sialylated galactose residues could be expressed in AFP upon malignant transformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-dimensional polyacrylamide gel electrophoresis of extracellular soybean pathogenesis-related proteins using PhastSystem

Acidic and basic pathogenesis-related proteins (PR-Ps) were extracted from the intercellular fluid (IF) of soybean leaves, locally infected with tobacco necrosis virus and showing necrotic local lesions. Proteins were detected by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using PhastSystem and precast commercially available gels. Extracts from healthy leaves were run as controls. PR-Ps were first run under native PAGE conditions or isoelectric focusing (IEF), the gels stained with Coomassie Blue, then run under sodium dodecyl sulfate (SDS)-denaturing conditions and finally stained with silver. Ten major acidic PR-Ps were separated; their Mr's were close to those found by conventional PAGE. Their isoelectric points ranged from 3.5 to 5.0. Ten basic PR-Ps were separated and their Mr's estimated. None of these acidic or basic soybean PR-Ps was a glycoprotein. PAGE with PhastSystem and precast gels gives reliable results, comparable with those from conventional 2D-PAGE, with simpler experimental procedures. By electrophoresing Coomassie-stained gels with SDS in the second dimension, we were able to control the first-dimensional separation and to avoid laborious protocols generally adopted with unstained gels.     

Two-dimensional electrophoresis of Cereus peruvianus (Cactaceae) callus tissue proteins

Two-dimensional electrophoresis of Cereus peruvianus callus tissues grown in culture media containing two different 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin combinations was used to identify minor differences in polypeptide composition of these cell clones. Altered expression during growth in the two 2,4-D and kinetin combinations was apparent for 13 polypeptides when calluses in the two media were compared. The number of proteins with differential expression (presence or absence of specific spots) was higher in callus tissues cultured in the 4.0 mg/L 2,4-D and 8.0 mg/L kinetin combination than in callus tissues cultured in the 4.0 mg/L 2,4-D and 4.0 mg/L kinetin combination. The present results show that the callus tissues maintained at 4.0 mg/L 2,4-D and 8.0 mg/L kinetin can be used as a matrix for in vitro selection programs.     

Two-dimensional electrophoresis of plasma proteins and high density lipoproteins during inflammation

Plasma protein and lipoprotein fractions of five patients were analyzed on day 1, 5, and 15 after severe head injury by combining three types of two-dimensional electrophoresis (2-DE) to obtain information on lipoprotein and apolipoprotein composition. On analysis under nondenaturing conditions in both dimensions on day 5, the samples show modifications of isoelectric point (pI) and molecular weight (Mr) properties of the high density lipoprotein (HDL) fraction in addition to an increase in inflammatory proteins and a return to a normal pattern on day 15. In the second type of 2-DE the samples were analyzed employing isoelectric focusing without denaturant in the first dimension, followed by sodium dodecyl sulfate (SDS) in the second dimension in order to study the protein composition of lipoprotein fractions. On day 5, a decrease of the apolipoproteins apo A-I, apo A-II, and apo C were noted, with simultaneous appearance of an unidentified protein with Mr 12,000 and pI 6.0. In the third type of 2-DE, employing urea and Nonidet P-40 in the first and SDS in the second dimension, the plasma polypeptide composition was studied. The presence of an unidentified polypeptide could be confirmed on day 5, tending to disappear thereafter. This Mr 12,000 component consists of two major spots at pI 5.7 and 6.0 and four minor ones between pI 6.0 and 8.0. These properties suggest that this protein corresponds to serum amyloid A apolipoprotein.     

Two-dimensional gel electrophoresis, protein electroblotting and microsequencing: a direct link between proteins and genes.

We have used two-dimensional gel electrophoresis as a general "preparative" method to purify proteins for microsequencing analysis. In the first experiments, proteins derived from a total extract of Nicotiana tabacum leaf tissue were directly blotted from the gel onto poly(4-vinyl-N-methylpyridinium iodide)-coated glass fiber sheets. The major spots were excised and subjected to NH2-terminal sequence analysis, which made it possible to identify five of the eight selected proteins, while two more were recognized by generated internal sequences. In a second set of experiments, proteins of human origin were separated on multiple two-dimensional gels and the Coomassie Brilliant Blue-stained spots were excised from the gels. The combined spots were re-eluted and concentrated in a new gel and blotted on Immobilon. They were fragmented by in situ proteolysis and the generated peptides were separated by reverse phase-high performance liquid chromatography and sequenced. At the average, the internal sequences that were obtained covered 35 residues per protein and allowed unambiguous identification of 13 of the 23 proteins analyzed so far. The sequence information obtained of the unidentified proteins is sufficient for further cloning. These results demonstrate that systematic sequence analysis of the major proteins seen in two-dimensional gels is within the reach of current technologies. This offers a unique opportunity to link information contained in protein databases with known or forthcoming DNA sequence data.     

Two-dimensional electrophoresis analysis of human serum proteins during the acute-phase response.

The serum of patients with meningitis, due to infection by Haemophilus influenzae type b, was analyzed. Several known acute-phase proteins were separated by two-dimensional electrophoresis and estimated quantitatively. In addition, hitherto undescribed reactants were recognized. Gels were calibrated and relevant spots related to master spot numbers in the human serum protein database.     

Two-dimensional electrophoresis and peptide mass fingerprinting of bacterial outer membrane proteins.

Many bacterial outer membrane proteins (OMPs) are missing from two-dimensional (2-D) gel proteome maps. Recently, we developed a technique for 2-D electrophoresis (2-DE) of Escherichia coli OMPs using alkaline pH incubation for isolation of OMPs, followed by improved solubilization conditions for array by 2-DE using immobilized pH gradients. In this report, we expanded our study, examining protein components from the outer membranes of two enteric bacteria, Salmonella typhimurium and Klebsiella pneumoniae (also known as Klebsiella aerogenes), as well as the unrelated, free-living alpha-proteobacteria Caulobacter crescentus. Patterns of OMPs expression appeared remarkably conserved between members of the Enterobacteriaceae, while C. crescentus was unique, displaying a greater number of clusters of higher-molecular-weight proteins (>80 kDa). Peptide mass fingerprinting (PMF) was used for protein identification, and despite matching across-species boundaries, proved useful for first-pass protein assignment of enteric OMPs. In contrast, identification of C. crescentus OMPs was successful only when searching against its recently completed genome. For all three microorganisms examined, the majority of proteins identified on the 2-D gel appear localized to the outer membrane, a result consistent with our previous finding in Escherichia coli. In addition, we discuss some of the benefits and limitations of PMF in cross-species searching.     

Two-dimensional gel electrophoresis as analytical tool for identifying Candida albicans immunogenic proteins

This paper reports the usefulness of two-dimensional gel electrophoresis followed by Western blotting with sera from patients with systemic candidiasis in the identification of the major Candida albicans antigens. In order to have different patterns of protein expression and subcellular localization, three types of protein preparations were obtained: cytoplasmic extracts, protoplast lysates and proteins secreted by protoplasts regenerating their cell wall. These proteins were separated by high-resolution two-dimensional electrophoresis using an immobilized pH gradient. Western blotting with sera from patients with systemic candidiasis allowed the detection of more than 18 immunoreactive proteins. Some of these proteins had different isoforms. All sera reacted with at least three C. albicans proteins and the most reactive serum detected up to eleven proteins. Some of these antigens, e.g., enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), have been identified on the 2-D map. The most reactive proteins were enolase and a 34 kDa protein in the acidic part of the gel (pI 4-4.4) that was only detected in regenerating protoplast-secreted proteins. The identification of all these antigens would be useful for the development of diagnostic strategies.     

Two-dimensional electrophoresis as a tool for structural and genetic studies of seed proteins from Poaceae and Fagaceae

The application of two-dimensional electrophoretic procedures to structural and genetic studies of seed proteins from Poaceae (including the cultivated cereals) and Fagaceae is described. The following related problems have been considered: covalent and non-covalent association of protein subunits in multiple oligomeric structures; chromosomal locations of genes encoding seed proteins; quantitation of gene products in relation to gene expression and regulation; purification of protein components to study their homology relationships and in vitro activities; evolutionary and phylogenetic relationships; identification of genetic stocks. Isoelectric focusing, pore-gradient electrophoresis, electrophoresis at different pH's, are among the separation procedures used in the first dimension, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis and starch-gel electrophoresis at acid pH have been the preferred second-dimensional methods. Dissociating conditions (sodium dodecyl sulfate, Nonidet P-40, or urea) and reducing conditions (2-mercaptoethanol) have been used when required.     

Two-dimensional electrophoresis aided by personal computer analysis for screening of mutant proteins in inherited diseases

A rapid and reproducible method of two-dimensional electrophoresis was developed for screening of abnormal proteins expressed in fibroblasts from patients with inherited diseases. After silver staining, the electrophoresis gel was subjected to semiautomatic digitizer-personal computer analysis: scanning with an image sensor video camera connected to a digitizer, followed by quantitative determination and statistical analysis with a personal computer. The protein spots analyzed by this method showed quantitative variations of various degrees, particularly in 2 of 247 spots examined. Seven spots were not always detected in control and pathological cells in this study. Slight variations in molecular weight were observed in 3 different spots.     

Two-dimensional polyacrylamide gel electrophoresis reveals differences between osteoblast and fibroblast extracellular proteins.

Normal human skin fibroblast primary cell lines secrete over 50 proteins into culture medium. These have been mapped previously using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and this technique has now been used to investigate extracellular protein secretion by human osteoblasts in vitro. We report the mapping of a number of consistent markers specific to the osteoblast. In particular, one protein chain with posttranslational modifications was found to be unique to the osteoblast extracellular protein map. The absence of the N- and O-glycoforms of collagenase from the osteoblast profile in this study concurs with findings reported using the immunoprecipitation functional assay and Northern blot analysis. The use of 2-D PAGE in phenotypic assessment provides a more complete analysis than the standard range of single-parameter tests for osteoblasts. Mapping of extracellular and cellular proteins in addition to bone matrix protein analysis will allow a comprehensive analysis of normal osteoblast function. This technique may also be applied to the study of osteoblasts in relation to bone disease and in assessing the phenotypic shift within a normal osteoblast culture.