Comparative study of electrophoretic patterns of latex proteins from clones of Hevea brasiliensis

Latex serum proteins from Hevea brasiliensis were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins from whole serum and fractions isolated by gel chromatography on Ultrogel AcA 44 were analyzed. No qualitative clonal differences were found in the protein patterns of whole latex or in the fractions but laser densitometry revealed reliable quantitative differences in protein composition. Reproducible mobilities and molecular weights of selected bands were obtained both within single gels as well as in different gels, analyzing several lots of latex received at various times from a Hevea experimental field station. The clones compared were IAN 710, GV 31, GV 42; the first one had the highest rubber yields.     

Analysis of membrane proteins by two-dimensional electrophoresis: comparison of the proteins extracted from normal or Plasmodium falciparum-infected erythrocyte ghosts

Parasite-encoded membrane proteins translocated to the surface of infected erythrocytes or in specialized vesicles underneath (Maurer's clefts) play a key role in the asexual life cycle of Plasmodium falciparum (a malaria-causing protozoan), by mediating key steps such as red blood cell invasion, sequestration of infected cells in microcapillaries, and red blood cell rupture. A large-scale analysis of these membrane proteins would therefore be of great help to gain knowledge of the different stages of the Plasmodium falciparum life cycle. In order to be able to detect and identify parasite-encoded proteins directed to the red blood cell membrane, we first defined the conditions required for optimal extraction and separation of normal red blood cell ghost proteins by two-dimensional gel electrophoresis. These conditions included the use of urea, thiourea and new zwitterionic detergents in the extraction and isoelectric focusing media. The optimized conditions were then applied to analyze normal and P. falciparum-infected red blood cell ghosts. Several protein spots were found only in infected ghosts and are expected to represent parasite-encoded proteins. These proteins are currently under investigation.     

Analysis of electrophoretic patterns of arbitrarily primed PCR profiling

We present a mathematical algorithm for the analysis of electrophoretic patterns resulting from arbitrarily primed PCR profiling. The algorithm is based on the established mathematical procedures applied to the analysis of digital images of gel patterns. The algorithm includes (a) transformation of the image into a matrix form, (b) identification of every electrophoretic lane as a set of matrix columns that are further mathematically processed, (c) averaging of matrix columns corresponding to electrophoretic lanes that define lane representatives, (d) elimination of "smiling" bands, (e) solving the problem of a lane offset, and (f) removal of the background. Representation of individual electrophoretic lanes in the form of functions allows interlane comparisons and further mathematical analysis. Direct comparison of selected lanes was obtained by employing correlation analysis. Gel images were those obtained after arbitrarily primed PCR analysis of DNA that underwent damage induced by gamma radiation from a (60)Co source. The applied method proved to be useful for elimination of subjectivity of visual inspection. It offers the possibility to avoid overlooking important differences in case of suboptimal electrophoretic resolution. In addition, higher precision is achieved in the assessment of quantitative differences due to better insight into experimental artifacts. These simple mathematical methods offer an open-type algorithm, i.e., this algorithm enables easy implementation of different parameters that may be useful for other analytical needs.     

Analysis of cyanobacterial pigments and proteins by electrophoretic and chromatographic methods

Cyanobacteria are a diverse and ubiquitous group of prokaryotes with several unifying features. Amongst these is the macromolecular structure known as the phycobilisome, which is composed of water-soluble phycobiliproteins covalently bound by linker peptides or proteins in a configuration designed to optimize energy transfer to the photosynthetic reaction center of the organism. Phycobiliproteins are highly fluorescent by virtue of their covalently bound, linear tetrapyrrole chromophores known as bilins. Analysis of these prosthetic pigments, along with other non-water soluble pigments, such as the chlorophylls and carotenoids, can provide insight into microbial diversity. The effects of environmental growth conditions and stresses can also be probed by measuring pigment and protein concentrations. This review will focus, therefore, on applications of various chromatographic and electrophoretic methods for the analysis of cyanobacterial pigment and protein constituents. Although the greatest emphasis will be placed on the measurement of bilins and phycobiliproteins, this review will also consider other pigments and proteins important to cyanobacterial growth and survival, such as chlorophyll a, carotenoids, ectoenzymes, linker and membrane proteins, and extracellular proteins.     

Two-dimensional electrophoretic analysis of nuclear proteins from human tumors

During the last decade several strategies have been developed to identify proteins which could serve as markers in tumor biology. One avenue of great promise to detect such proteins seems to be the separation of prefractionated organelles from tumor cells by high resolution two-dimensional electrophoresis. Using detergent-lysed nuclei from several human tumor cell lines, especially from brain tumors, and two-dimensional electrophoresis, we analyzed the nuclear protein pattern obtained after sequential salt extraction of tumor cell nuclei. In addition to proteins occurring in all tumor cell lines, the pattern of different tumor cell lines exhibits considerable differences when proteins were visualized by silver staining, thus emphasizing the specificity of nuclear proteins with respect to the cell type. Even quantitative variations of the nuclear phosphoproteins 23/4 were detectable, indicating a potential correlation between their synthesis/phosphorylation and the proliferation behavior of tumor cells. The data indicate that nuclear proteins with their distinct heterogeneity and tissue specificity may represent a powerful source in determining tumor-specific proteins. The extent of chromosomal protein heterogeneity may be additionally increased by their covalent modification by nuclear kinases; therefore, tumor-specific nuclear proteins may occur as quantitative and qualitative variations.     

Analysis of similarity of electrophoretic patterns in mRNA differential display

We present a novel method of statistical analysis for the comparison of electrophoretic data. The method is based on the squared Euclidian distance of normalized signal data vectors of electrophoretic lanes. The differences in the electrophoretic patterns are evaluated by a statistical test based on Hubert's statistics which measures the significance of the signal grouping. We demonstrate the validity and applicability of the method in a large data set derived from automated fluorescent mRNA differential display analysis of the expression of acute-phase proteins during experimental Escherichia coli infection in mice. The current testing method is capable of finding theoretically similar natural groupings to be similar in a statistically significant way whereas theoretically dissimilar or random groupings can be recognized to be artifactual. We also show how the calculated pairwise signal distances can be utilized in methodological problem solving. These analytical methods can be applied to the study of other related problems of similarity analysis of electrophoretic patterns, and also provide useful tools for the development of automated recognition of differentially expressed mRNAs.     

Generation of minimal protein identifiers of proteins from two-dimensional gels and recombinant proteins

We describe the technical feasibility and methodology to characterize a protein by a minimal set of structural information generated by matrix assisted laser desorption/ionization (MALDI)-mass spectrometry, termed a "minimal protein Identifier" (MPI). MPIs can be determined for proteins from two-dimensional gels and recombinant proteins and can be used to compare and identify proteins from these sources.     

Characterization of antithrombin III from human plasma by two-dimensional gel electrophoresis and capillary electrophoretic methods

The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and pI differences down to 0.01 pH units could be distinguished. ATIII-alpha and ATIII-beta-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-alpha and ATIII-beta exhibit pI values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The pI difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-beta. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-alpha main isoform and about 6.6% of ATIII-beta. The pI values of ATIII determined by CIEF with internal calibration were in fair agreement with the pI values of the main isoforms achieved with 2-DE.     

Analysis of human tear proteins by two-dimensional electrophoresis.

Human tear proteins in the conjunctival sac were separated on the basis of the differences in their isoelectric points and molecular weights using micro two-dimensional electrophoresis combined with immunoblotting. The two-dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis were compared with those from normal individuals. We also measured integrated intensities of seven protein spots, lactoferrin (LF), albumin and five specific tear proteins (STP), to examine differences in the amounts of these proteins in tears from normal individuals of different sexes. In the tears from patients with conjunctivitis, secretory immunoglobulin A (IgA), LF and STP spots were stained more weakly, whereas the albumin spot was stained more strongly as compared with those from normal individuals. Furthermore, haptoglobin and IgG spots appeared in the tears from patients with conjunctivitis. These were more prominent in the tears from patients with severe conjunctivitis. There were significant differences in the amounts of LF and two kinds of STPs in the different sexes. The amounts of these proteins were larger in females.     

Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis

The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)-anchored proteins y two-dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent-resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI-anchored proteins. Although the GPI-anchored proteins were readily separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these proteins were undetectable on two-dimensional (2-D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent-resistant membranes such as caveolin-1 and flotillin-1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI-anchored proteins required cleavage of the lipid tail with phosphatidylinositol-specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI-anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI-anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N-glycosylation. Pretreatment with N-glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N-linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray-tandem mass spectrometry as illustrated for the GPI-anchored protein, Thy-1.     

Distortion of the electrophoretic titration curves of some proteins

The electrophoretic titration curves of complex mixtures of vitamin K-dependent human blood proteins and proteins of Bothrops asper venom were investigated. In both protein mixtures some curves exhibited marked distortions such as additional maxima and minima when Pharmalyte 3-10 carrier ampholytes were used for isoelectric focusing in agarose gels. The distortions result from an unspecific interactions between some carrier ampholyte constituents with particular proteins. The interacting carrier ampholyte components could be completely removed by binding to albumin and ultrafiltration through a UM-2 Amicon membrane with resultant regular titration curves. The interacting carrier ampholyte species were only partially removed by ultrafiltration through a UM-2 membrane without incubation with albumin.     

Comparison of two-dimensional electrophoresis patterns of heat shock protein Hsp27 species in normal and cardiomyopathic hearts

Heat shock protein Hsp27 occurs in a complex pattern in human myocardial tissue. Normal and failing explanted human heart from patients with dilated cardiomyopathy (DCM) or ischemic heart failure (IHF), respectively, were analyzed by high resolution two-dimensional electrophoresis (23x30 cm) and Hsp27 immunostaining. Twelve Hsp27 spots in DCM samples were significantly altered in intensity and ten of these were significantly changed in IHF. Four spots (h1, h2, h4, h5) in DCM samples and three spots (h2, h4, h5) in IHF at a molecular mass of 28 kDa were decreased in intensity. In this study, investigating left ventricles of human myocardium, spot h4 was only detected in normal heart samples. On the other hand, spots with a lower molecular mass of 27 kDa (h14, h15, h17, h20, h21) and 22-23 kDa (46, h47, h50) increased in intensity in failing hearts, suggesting that some form of Hsp27 degradation occurs during heart failure.     

Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis

To increase the number of proteins detectable by two-dimensional electrophoresis (2-DE) in plants, we present a new procedure for extracting total proteins from plant tissue. This method avoids any loss of proteins in the course of sample preparation and results in two different fractions, one comprising mainly the cytoplasmatic proteins, the other one containing predominantly structure bond proteins. 2-DE patterns obtained from these two fractions show that the total number of different protein spots detected exceeds the degree of resolution commonly reported for plant proteins threefold.     

Microsequences of 145 proteins recorded in the two-dimensional gel protein database of normal human epidermal keratinocytes.

Microsequencing of proteins recovered from two-dimensional (2-D) gels is being used systematically to identify proteins in the master human keratinocyte 2-D gel database. To date, about 250 protein spots recorded in human 2-D gel databases have been microsequenced and, of these, 145 are recorded in the keratinocyte database under the entry partial amino acid sequence. Coomassie Brilliant Blue-stained protein spots cut from several (up to 40) dry gels were concentrated by elution-concentration gel electrophoresis, electroblotted onto PVDF membranes and digested in situ with trypsin. Eluting peptides were separated by reversed-phase HPLC, collected individually and sequenced. Computer search using the FASTA and TFASTA programs from Genetics Computer Group indicated that 110 of the microsequenced polypeptides shared significant similarity with proteins contained in the PIR, Mipsx or GenEMBL databases. Only 35 polypeptides corresponded to hitherto unknown proteins. Peptide sequences of all 145 proteins are listed together with their coordinates (apparent molecular weight and pI) in the keratinocyte database.     

Exploring the evaluation of net charge, hydrodynamic size and shape of peptides through experimental electrophoretic mobilities obtained from CZE

This work explores the validity of simple CZE models to analyze the electrophoretic mobilities of 102 peptides reported in literature. These models are based mainly on fundamental physicochemical theories providing analytical expressions amenable to relatively simple numerical analysis. Thus, the Linderstr?m-Lang capillary electrophoresis model (LLCEM) and its perturbed version (PLLCEM), proposed and applied previously to the CZE of globular proteins, are adapted and used here for peptides. Also the effects of pK-shifts on net charge, hydration and hydrodynamic size and shape of peptides are analyzed and discussed. Emphasis is placed on the fact that these parameters are physically coupled, and thus a variation in the net charge may produce an appreciable change in the hydrodynamic size of peptides. Within the framework of CZE, peptides may be assumed as having a hydrodynamic volume associated with either spherical or spheroidal particles. The effects on peptide net charge and hydrodynamic size, of electrostatic interaction between a pair of charged groups in the chain and electrical permitivitty around the peptide domain are studied. The predictions of the PLLCEM and LLCEM are in good agreement with results reported previously in the literature. Several limitations concerning these models and some needs for further research are also described.     

Identification of immunoreactive proteins of Chlamydia trachomatis by Western blot analysis of a two-dimensional electrophoresis map with patient sera.

Western blots of two-dimensional electrophoretic maps of proteins from Chlamydia trachomatis were probed with sera from 17 seropositive patients with genital inflammatory disease. Immunoblot patterns (comprising 28 to 2 spots, average 14.8) were different for each patient; however, antibodies against a spot-cluster due to the chlamydia-specific antigen outer membrane protein-2 (OMP2) were observed in all sera. The next most frequent group of antibodies (15/17; 88%) recognized the hsp60 GroEL-like protein, described as immunopathogenic in chlamydial infections. Reactivity to the major surface-exposed and variable antigen major outer membrane protein (MOMP) was observed at a relatively lower frequency (13/17; 76%). The hsp70 DnaK-like protein was also frequently recognized (11/17; 64.7%) in this patient group. Besides the above confirmatory findings, the study detected several new immunoreactive proteins, with frequencies ranging from 11/17 to 1/17. Some were characterized also by N-terminal amino acid sequencing and homology searches. Amongst these were a novel outer membrane protein (OmpB) and, interestingly, five conserved bacterial proteins: four (23%) sera reacted with the RNA polymerase alpha-subunit, five (29%) recognized the ribosomal protein S1, eight (47%) the protein elongation factor EF-Tu, seven (41%) a putative stress-induced protease of the HtrA family, and seven sera (41%) the ribosomal protein L7/L12. Homologs of the last two proteins were shown to confer protective immunity in other bacterial infections. The data show that immunological sensitization processes commonly thought to play a role in chlamydial pathogenicity may be sustained not only by the hsp60 GroEl-like protein, but also by other conserved bacterial antigens, some of which may be also considered as potential vaccine candidates.     

Reference map of soluble proteins from Streptococcus thermophilus by two-dimensional electrophoresis

Streptococcus thermophilus is a lactic acid bacterium widely used for the production of fermented dairy products. The two-dimensional electrophoresis (2-DE) protein profile was obtained from three independent analyses of 2-DE gels of soluble proteins of the strain PB18. About 270 spots were detected by silver staining and the average molecular weight and isoelectric point of each protein spot were calculated to be 41 600 and 5.2, respectively. Twelve proteins were purified by chromatographic techniques because their concentration was too low for direct sequencing from blots. Eleven were located in the PB18 2-DE profile after silver staining. These preliminary results contribute to the setting up of a two-dimensional image (or reference map) of the proteins from S. thermophilus in order to identify and compare strains of various origin or to follow metabolic process such as stress. Bidimensional autoradiographs of two strains (PB18 and ST105) of S. thermophilus grown in exponential phase at 42 degrees C with [35S]methionine were compared with an image analysis system. Among the eleven located proteins in the 2-DE silver-stained profile, nine were found in PB18 and eight in ST105 autoradiographs. One protein was specific to PB18. The eight proteins could play the role of internal 2-D PAGE markers of p/ and Mr for S. thermophilus.