Characterization of the major proteins of bovine seminal fluid by two-dimensional polyacrylamide gel electrophoresis

Recently, we demonstrated that the major proteins from bovine seminal plasma BSP-A1, -A2, -A3 and -30-kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5-7.8 and at molecular weights (M(r)) between 6 and 100 kDa. isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D-PAGE. BSP-A1 was found at a M(r) of 16.5 kDa and in the range of pl of 4.7-5.0; BSP-A2 at 16 kDa and at a pl of 4.9-5.2; BSP-A3 at 15 kDa and at a pl of 4.8-5.2, and BSP-30-kDa at 28 kDa and at a pl of 3.9-4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP-30-kDa to 4.8-5.0 and decreased its M(r) to 25 kDa, but no change was observed for BSP-A1, -A2 and -A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their M(r) nor their pl. Furthermore, when CBSP proteins were separated in 2D-PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP-30-kDa is mainly due to its sialic acid content.     

Agarose gel electrophoresis of cerebrospinal fluid proteins and analysis of the pherogram profiles by analog computer

A micro-method of agarose gel electrophoresis requiring 1--3 ml of cerebrospinal fluid for quantitative analysis of cerebrospinal fluid proteins is described. After concentration of CSF to about 50 microliter by ultrafiltration and refractometric determination of protein, approximately 20--40 microgram of total protein are used for electrophoresis. Photometric scanning of the electrophoretic pattern at two different wavelengths permits quantitative evaluation. The pherograms are analysed by means of a modified DU-PONT analog computer. Factors which influence quantitative electrophoresis are examined. In cerebrospinal fluid of normal children 15 protein fractions are demonstrated: 2 prealbumins, albumin, 5 alpha-, 3 beta- and 4 gamma-globulins.     

S-100 protein concentration in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease

We evaluated S-100 levels in paired cerebrospinal fluid (CSF) and serum samples in a group of 135 patients referred to the German Creutzfeldt-Jakob disease (CJD) surveillance unit from June 1993 to May 1995. The patients were seen in a prospective case control study. The diagnosis of probable CJD during life was made in any patient presenting with rapidly progressive dementia of less than 2 years' duration, typical periodic sharp wave complexes (PSWCs) in the EEG and at least two of the following findings: myoclonus, visual/or cerebellar symptoms, pyramidal and/or extrapyramidal signs and/or akinetic mutism. Patients presenting with the above clinical signs and symptoms but without PSWCs were classified as possible, while those with a dementia of a duration exceeding 2 years and without PSWCs were classified as other. S-100 was determined in paired CSF and serum samples by a commercially available enzyme-linked immunosorbent assay. In a group of 76 patients with definite and probable CJD, S-100 concentration (median 25 ng/ml, range 2-117) in CSF was significantly higher (P < 0.0001) than in 32 patients diagnosed as other (median 4 ng/ml, range 1-19). Serum levels of S-100 were below 0.5 ng/ml in all groups. At a cut-off of 8 ng/ml an optimum sensitivity of 84.2% with a specificity of 90.6% for the diagnosis of CJD by the determination of S-100 in CSF is obtained. S-100 levels exceeding 8 ng/ml in CSF support the diagnosis of CJD in any patient presenting with rapidly progressive dementia.     

Preparative two-dimensional gel electrophoresis of membrane proteins

Electrophoretic techniques, and especially two-dimensional gel electrophoresis (2-DE), have provided an indispensable set of tools for the separation of complex protein mixtures as well as for the identification of protein-protein interactions. Nevertheless, after its introduction more than twenty years ago and even with recent technical developments, the separation of integral and peripheral membrane proteins, in amounts sufficient for microsequencing, is still a difficult task. Lipids present in the membrane as well as the low solubility of hydrophobic membrane proteins result in protein aggregation both on the sample application point and on isoelectric focusing. As a consequence many proteins do not enter the first or second dimension of 2-DE. Here we describe the modification of a protocol using a combination of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), chaotropic agents (thiourea, urea), Tris base and reducing agents (1,4-dithioerythritol) to improve solubilization of integral and peripheral membrane proteins. Preparative amounts of membrane proteins (up to 2 mg) were loaded during reswelling of dry immobilized pH gradients and the resulting Coomassie staining patterns were largely superimposable with silver-stained gels obtained from identical samples (4 microg). This indicates that the recovery of proteins from the sample is not significantly compromised by the scale-up procedure. A direct application of this method for the characterization and identification of membrane proteins from cellular organelles is described in another paper in this issue (I. Fialka et al., Electrophoresis 1997, 18, 2582-2590).     

Protein analysis of Strongyloides venezuelensis by two-dimensional polyacrylamide gel electrophoresis

Infective larvae, larvae in the lung and adult-stage worms in the small intestine of Strongyloides venezuelensis were analysed for protein by two-dimensional gel electrophoresis. The infective larvae were differentiated from the other two stages of parasite with 13 stage-specific spots, whereas the larvae in the lung and the adult-stage worms were identical to each other in spot patterns except for 6 spots.     

Apolipoprotein E phenotype frequency and cerebrospinal fluid concentration are not associated with Creutzfeldt-Jakob disease

OBJECTIVE: To develop a new outcome measure in response to the increasing demands for cost effectiveness analyses and empirically derived outcome instruments in the treatment of pediatric limb deficiency. This article describes the development, refinement, and initial psychometric properties of the Child Amputee Prosthetics Project-Functional Status Inventory (CAPP-FSI). DESIGN: Parents of children with limb deficiency were surveyed during routine clinic visits. SETTING: Two outpatient pediatric clinics. PARTICIPANTS: Seventy-five parents and their children with limb deficiency (ages 8 to 17 years) participated in the study as part of annual physical evaluations or routine follow-up care. MAIN OUTCOME MEASURE: The newly developed CAPP-FSI. RESULTS: Internal consistency reliability (Cronbach's alpha) = .96 for the CAPP-FSI. Content validity is described and initial construct validity is empirically confirmed. CONCLUSION: The CAPP-FSI is a promising assessment instrument for measuring important health outcomes in children with upper or lower limb deficiency.     

Correlation of plasma protein separation patterns obtained by two-dimensional polyacrylamide gel electrophoresis and by capillary electrophoresis

We used three different electrophoretic techniques for the analysis of human plasma proteins: (i) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), with sodium dodecyl sulfate (SDS) used only in slab gel electrophoresis; (ii) capillary isoelectric focusing (CIEF) with no denaturants; (iii) linear polyacrylamide (LPA)-filled capillary electrophoresis with SDS (SDS-CE). With technique (i), data on isoelectric point and molecular size of plasma proteins can be obtained. Techniques (ii) and (iii) are suited to obtain quantitative information on proteins. The separation principle used in technique (ii) is closely related to that used in the first dimension of technique (i), and that used in technique (iii) related to that in the second dimension of technique (i). Therefore, we could successfully correlate protein separation patterns obtained by 2-D PAGE and those obtained by capillary electrophoresis. The advantages of correlating data obtained by various electrophoretic techniques in the course of constructing a comprehensive database on human plasma proteins are discussed.     

Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis

For identification of Rab, Rac, Rho, Ral, Rap, and Arf proteins on two-dimensional polyacrylamide gels, we have expressed full-length cDNAs of members of these protein families with the T7 RNA polymerase-recombinant vaccinia virus expression system. Membrane preparations from cells expressing the cDNAs were subjected to high-resolution two-dimensional polyacrylamide gel electrophoresis followed by [alpha-32P]GTP ligand blotting. We have mapped 28 small GTP-binding proteins relative to their isoelectric points and according to their molecular weights and by immunoblotting with specific antibodies. Rab and Rho proteins could be specifically identified by extraction of streptolysin O-permeabilized Madin-Darby canine kidney (MDCK) cells with Rab- and Rho-GDP dissociation inhibitor. We applied the reference mapping to analyze the GTP-binding patterns of synaptosome fractions from rat brain. The purified synaptosomes exhibited specific enrichment of Rab3a, Rab5a, Ral, and several other GTPases. This approach and the map we have produced should provide a useful aid for the analysis of the expression and localization of members of all families of small GTP-binding proteins in various cell types and subcellular fractions.     

Clinical implications of the types of cryoglobulins determined by two-dimensional polyacrylamide gel electrophoresis

BACKGROUND AND OBJECTIVE: Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a new method which can be used to study cryoprecipitates from the sera of cryoglobulinemic patients. It led to the identification of a new type of cryoprecipitate, tentatively named II-III, characterized by polyclonal IgG associated with a mixture of polyclonal and monoclonal IgM. Some discrepancies with the conventional classification of cryoglobulins were revealed. The association of particular clinical features with the classification of cryoglobulins by 2-D PAGE is examined. DESIGN AND METHODS: Sixty consecutive patients affected by cryoglobulinemic syndrome with mixed cryoglobulins were included in the study. All patients were evaluated for cutaneous, articular, hepatic, renal and nervous involvement. The washed cryoprecipitates were typed using both techniques: immunofixation electrophoresis (IFE) and 2-D PAGE. RESULTS: Sixteen (6 cases of type II and 10 of type III by IFE) of 60 cryoprecipitates (26.6%) appeared as type II-III by 2-D PAGE analysis. Nine cases were classified differently by IFE and 2-D PAGE. Mixed cryoglobulins of type II-III were not associated with a particular clinical pattern. Examining the clinical findings in the mono group (those with monoclonal IgM alone) and the poly group (those with polyclonal IgM alone or polyclonal and monoclonal IgM) we found clearly significant differences: more severe liver involvement in the poly group, and higher cryocrit and creatinine values, lower C4 level and more severe purpura in the mono group. INTERPRETATION AND CONCLUSIONS: Our results confirm the reliability of 2-D PAGE in characterizing cryoprecipitates. This sensitive method can demonstrate a higher number of monoclonal components, undetectable by IFE. Type II-III cryoglobulins are not associated with a particular clinical pattern. The presence or absence of polyclonal IgM in mixed cryoglobulins seems to be correlated with some clinical findings.     

Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis

We describe the extraction and enrichment of membrane proteins for separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three-step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris-base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N-terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2-D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2-D PAGE using broad-range pH 3.5-10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.     

The interaction between Mycobacterium and the macrophage analyzed by two-dimensional polyacrylamide gel electrophoresis

The intramacrophage pathogen Mycobacterium avium resides in a vacuole which displays unusual fusion characteristics, expressed as both a failure to mature into phagolysosomes and a continued access to the early recycling pathway. In contrast, compartments containing inert IgG-opsonized latex beads mature to phagolysosomes. Techniques were developed for the isolation of these particle-containing phagosomes from macrophages to facilitate analysis of phagosomal constituents by electrophoresis and autoradiography. Metabolic labeling of macrophages followed by phagosome isolation and two-dimensional polyacrylamide gel electrophoresis revealed only minor differences in the protein profiles between the M. avium and IgG-bead phagosomes despite the marked differences in the fusigenicity of the respective vacuoles. Pulse-chase labeling experiments revealed greater differences in the accessibility of Mycobacterium avium and IgG-bead phagosomes to newly synthesized proteins. These phagosome isolation techniques were extended to analyze the protein synthesis profile of intracellular M. avium for comparison with bacteria that were metabolically labeled in broth culture. Not surprisingly, the majority of polypeptides in the bacilli were common to both growth conditions. However, despite these similarities, intracellular M. avium express several unique proteins, most notably one abundant protein with a molecular weight of 51 kDa. In addition, the bacteria manifest a restricted set of proteins expressed while in stasis shortly after infection.     

Diagnosis of meningococcal meningitis by broad-range bacterial PCR with cerebrospinal fluid

We used broad-range bacterial PCR combined with DNA sequencing to examine prospectively cerebrospinal fluid (CSF) samples from patients with suspected meningitis. Fifty-six CSF samples from 46 patients were studied during the year 1995. Genes coding for bacterial 16S and/or 23S rRNA genes could be amplified from the CSF samples from five patients with a clinical picture consistent with acute bacterial meningitis. For these patients, the sequenced PCR product shared 98.3 to 100% homology with the Neisseria meningitidis sequence. For one patient, the diagnosis was initially made by PCR alone. Of the remaining 51 CSF samples, for 50 (98.0%) samples the negative PCR findings were in accordance with the negative findings by bacterial culture and Gram staining, as well as with the eventual clinical diagnosis for the patient. However, the PCR test failed to detect the bacterial rRNA gene in one CSF sample, the culture of which yielded Listeria monocytogenes. These results invite new research efforts to be focused on the application of PCR with broad-range bacterial primers to improve the etiologic diagnosis of bacterial meningitis. In a clinical setting, Gram staining and bacterial culture still remain the cornerstones of diagnosis.     

Development of a two-dimensional gel electrophoresis database of human lysosomal proteins

Two-dimensional gel electrophoresis databases have been generated for a range of tissue cell and fluid types, from a number of species. A major difficulty in the development of such databases is the large number of proteins present in even a single cell type (> 10,000) and the low levels of many of these proteins. One approach to overcome these difficulties is to fractionate the cell into its organelles and generate individual databases for each subcellular component. This has the added advantage of assigning a cellular location to each protein identified. Here we report the development of a two-dimensional gel electrophoresis database of lysosomal proteins.     

A fast spot segmentation algorithm for two-dimensional gel electrophoresis analysis

An important issue in the automation of two-dimensional gel electrophoresis image analysis is the detection and quantification of protein spots. A spot segmentation algorithm must detect, define the extent of, and measure the integrated density of spots under a wide variety of actual gel image conditions. Besides these functions, the algorithm must be memory efficient to be able to process very large gel images and do this in a reasonable amount of computation time on low-cost computers, such as workstations and personal computers. We have developed a fast spot segmentation algorithm, extending the GELLAB-II segmenter, which extracts spots in a single raster scanning pass through the gel image. The performance analysis of the algorithm will be given in the paper as well as a discussion of the algorithm.     

Characterization of proteins from human cerebrospinal fluid by a combination of preparative two-dimensional liquid-phase electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

To purify and characterize low-abundance proteins in complex biological mixtures, we used a novel strategy that combined preparative two-dimensional liquid-phase electrophoresis (2D-LPE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Preparative 2D-LPE is based on the same isoelectric focusing and gel electrophoresis principles as the widely used analytical 2D gel electrophoresis, except that analytes remain in solution throughout separation. This novel approach shows many improvements compared to analytical 2D gel electrophoresis for the separation of proteins in biological fluids. For example, larger volumes/amounts of samples can be loaded, yielding sufficient amounts of low-abundance proteins for further characterization. Since proteins remain in liquid phase during the entire procedure, extra steps such as electroelution, extraction, or transfer to membranes from the gels prior to mass spectrometric analysis are obviated. We report the usefulness of 2D-LPE combined with MALDI-TOF MS for the purification and characterization of cystatin C and beta-2 microglobulin in human cerebrospinal fluid. This method should be applicable to a wide range of biological fluids, such as cerebrospinal fluid, serum, tissue extracts, cell media, whole cells, and bacterial lysates.     

A computer model for the analysis of DNA replication intermediates by two-dimensional agarose gel electrophoresis

We present a computer model to predict the patterns expected for the replication intermediates (RIs) of DNA fragments analyzed by neutral/neutral two-dimensional (2D) agarose gel electrophoresis. The model relies on the mode of replication (uni- or bi-directional), the electrophoretic mobility of linear DNA fragments and the retardation caused by the three-dimensional shape of non-linear molecules. The utility of this model is demonstrated with two examples: replication analysis of the plasmids pBR322 and pHH5.8 in Escherichia coli after digestions with EcoRI and HindIII, respectively.     

Effects of renovascular hypertension on myocardial protein patterns: analysis by computer-assisted two-dimensional gel electrophoresis

Hypertensive heart disease caused by renovascular hypertension reflects the response of the heart to an increased afterload and neurohormonal stimulation. We hypothesized that in this condition the composition of the myocardial proteins of rats was altered. To identify yet unknown quantitative and qualitative differences in myocardial proteins in renovascular hypertensive heart disease, we analyzed protein patterns by computer-assisted two-dimensional polyacrylamide large gel electrophoresis. Renovascular hypertension was induced by placing a silver clip on the left renal artery in 9-week-old rat siblings. Sham-operated animals served as controls. Systolic blood pressure (197 +/- 19 mm Hg) and heart/body weight ratios (0.36 +/- 0.04%) were significantly increased in the hypertensive animals. Twenty protein patterns from the left ventricle of five hypertensive and five control rats were compared. The molecular mass and isoelectric point (pI) of proteins spots ranging from 13 to 100 kDa and from 4.5 to 8.5, respectively, were determined using marker proteins. In total, 761 +/- 88 protein spots were resolved in all twenty gels. For the quantitative data analysis a univariate (Mann-Whitney test) as well as a multivariate statistical approach (correspondence analysis) were applied. Only one myocardial protein spot (molecular mass = 41.3 kDa; pI = 6.3) was decreased by more than twofold (p < 0.05) in renovascular hypertension. The vast majority of spots did not indicate a significant alteration of intensity. Left ventricular hypertrophy in early renovascular hypertension induces a form of myocardial hypertrophy that conserves the naturally occurring protein expression pattern.     

Topological complexity of different populations of pBR322 as visualized by two-dimensional agarose gel electrophoresis

Neutral/neutral two-dimensional (2D) agarose gelelectrophoresis was used to investigate populations of the different topological conformations that pBR322 can adopt in vivo in bacterial cells as well as in Xenopus egg extracts. To help in interpretation and identification of all the different signals, undigested as well as DNA samples pretreated with DNase I, topoisomerase I and topoisomerase II were analyzed. The second dimension of the 2D gel system was run with or without ethidium bromide to account for any possible changes in the migration behavior of DNA molecules caused by intercalation of this planar agent. Finally, DNA samples were isolated from a recA-strain of Escherichia coli , as well as after direct labeling of the replication intermediates in extracts of Xenopus laevis eggs. Altogether, the results obtained demonstrated that 2D gels can be readily used to identify most of the complex topological populations that circular molecules can adopt in vivo in both bacteria and eukaryotic cells.