The apoE polymorphism studied by two-dimensional, high-resolution gel electrophoresis of serum

The apoE polymorphism of human very low density lipoprotein (VLDL) was studied by the two-dimensional high-resolution gel electrophoresis technique of O'Farrell, which combines isoelectric focusing and SDS-polyacrylamide gel electrophoresis. With whole serum, six major patterns and two "sub-variants" of apoE were observed. A series of twin pairs (21 monozygotic (MZ) and 13 dizygotic (DZ) pairs) as well as unrelated people were analyzed. Both members of MZ pairs always exhibited the same pattern, whereas DZ twins were often discordant. The patterns observed would be consistent with the hypothesis that three apoE isopeptides are coded for by three different alleles at one single locus. In this small series, all three postulated homozygous patterns, namely apoE-II, apoE-III and apoE-IV as well as the three heterozygous patterns apoE-11, 111; apoE-III, IV and apoE-ll, IV were seen.     

Interaction of heparinized polymer materials with plasma proteins and platelets

Conclusion A negative correlation was found between adsorption of plasma proteins and surface affinity for AT-III. This correlation suggests that maximal activity of surface-bound HP and, therefore, maximal hemocompatibility of polymer material can be attained by minimization of adsorbed plasma proteins. The adsorbed layer is also enriched with AT-III. In addition, a negative correlation in the AT-III-RNAP pair shows that this condition is also necessary for decreasing risk of thrombosis, because it reduces the number of adhered platelets.Thus, high concentration of AT-III in the adsorbed layer is the specific feature of heparinized biomaterials which exerts a fundamental effect on cellular and molecular mechanisms of the interaction of blood with polymer materials.Scientific-Research Institute for Transplantology and Artificial Organs, Russian Ministry of Health, Moscow. Translated from Meditsinskaya Tekhnika, No. 2, pp. 18–22, March–April, 1994.     

Mapping of seminal plasma proteins by two-dimensional gel electrophoresis in men with normal and impaired spermatogenesis

The present study analyses differential polypeptide expression of seminal plasma from fertile and infertile men by two-dimensional gel electrophoresis. Optimization of solubilization of seminal plasma was obtained by using [3-(3-(cholamidopropyl) dimethyl-ammonio)-1-propane sulphonate] and chaotropic agent mixture in lysis buffer before separation in immobilized pH gradient for isoelectric focusing. A two-dimensional map of seminal plasma from a fertile man allowed the detection of about 750 spots. Semi-preparative electrofocusing was performed. Analysis of tryptic fragments of two major spots by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectroscopy resulted in identification of prostatic acid phosphatase and prostate specific antigen. Three groups of spots and seven individual spots of isoelectric point from 4.6 to 6.2 and mol. wt from 41 to 18 kDa disappeared in the two-dimensional maps of seminal plasma of vasectomized men (n = 4) and of a patient with bilateral anorchidy as compared to that of fertile men (n = 5). Some of these polypeptides were also absent in seminal plasma of patients with alterations of seminiferous tubules showing Sertoli cell-only syndrome characteristics (n = 4) and could be potential diagnostic markers of spermatogenesis impairment.     

Identification of isolate-specific sporozoite proteins of Cryptosporidium parvum by two-dimensional gel electrophoresis.

Five isolates of Cryptosporidium parvum collected from human, horse, and calf sources were compared for differences in sporozoite protein patterns by using two-dimensional gel electrophoresis. Silver-stained two-dimensional gels contained over 300 protein spots from detergent-solubilized sporozoites. A distinguishing 106-kilodalton peptide that shifted in isoelectric point was detected in four of the five isolates. Computerized two-dimensional gel analysis was performed to obtain objective quantitation of the pI shift. Three of these four isolates could be differentiated from one other by the pI shift in this peptide. The fifth isolate was distinguished by the absence of the 106-kilodalton peptide and the presence of a 40-kilodalton peptide that was not observed in any other isolate.     

Development of monoclonal antibodies to proteins separated by two-dimensional gel electrophoresis

We have developed techniques for the production of monoclonal antibodies using Coomassie blue-stained protein spots cut from high resolution 2-dimensional polyacrylamide gels. The gel spots were homogenized with Freund's adjuvant and injected sub-cutaneously into a mouse, at several places along the flank. After boosting twice the spleen cells were hybridized by standard methods. Hybrids, clones and ascitic fluids were also screened with antigen prepared from 2-dimensional gel spots. The spots were cut from gels, homogenized in the presence of guanidinium chloride, and extracted by shaking overnight. The acrylamide was removed, the sample dialyzed to remove denaturant and the protein labeled with 125I. An alternative method for the production of screening antigen using column chromatography is described. These techniques allow the production of monoclonal antibodies to specific protein components of complex mixtures, even in the presence of other immunodominant proteins.     

Expressed immunoglobulin repertoire of LPS-stimulated splenocytes of unimmunized mice as studied by two-dimensional gel electrophoresis.

The repertoire of isolated immunoglobulin polypeptide chains synthesized by LPS-stimulated splenic B cells from unimmunized 6 weeks old mice was studied by two-dimensional gel electrophoresis. These B cells formed mainly mu heavy chains, while only a small amount of gamma chains was detected on two-dimensional electrophoregrams. The number and character of spots corresponding to each class and type of H and L chains were analyzed. Most of the detected 52 spots, which corresponded to L chains, were well resolved with clearly defined round boundaries. Six of them belonged to two isotypes of lambda chains and the rest to the kappa chain. About 25 clusters corresponded to mu chains. They had different appearance from those of L chains and their characteristic elliptic form with prolonged vertical axes indicated the presence of several H chain variants of slightly different length (due probably to the length variations of CDR3 and carbohydrate heterogeneity) in each cluster. The limited number of spots both of H and L chains is explained as being due to restrictions in the expressed repertoire of preimmune splenic B cells, which have no somatic mutations in the immunoglobulin genes. The concept of macrorepertoire (referring to the relatively small number of detected molecular species) and microrepertoire (describing the mutationally altered molecules) is introduced.     

Comparison of synaptic plasma membrane and synaptic vesicle polypeptides by two-dimensional polyacrylamide gel electrophoresis.

Extracts of chick brain synaptic plasma membranes, synaptic vesicles, and mixtures of membranes and vesicles were examined by electrophoresis on two-dimensional polyacrylamide gels by a modification of the O'Farrell technique. Synaptic plasma membranes had twenty-one major polypeptides; synaptic vesicles had seventeen. Thirteen major polypeptides were common to both fractions. The similarities between the synaptic vesicle and synaptic plasma membrane patterns are unlikely to be due to contamination of one fraction by the other or to contamination of both fractions by microsomes, synaptoplasm or mitochondria. Our findings are consistent with mixing of membrane proteins occurring during exocytosis but it remains to be shown that these synaptic subfractions are not contaminated by a type of membrane for which markers are not yet available.     

Comparative analysis of Trichinella spiralis and Trichinella nativa proteins by two-dimensional gel electrophoresis

Trichinella spiralis and Trichinella nativa are both common wildlife parasites in Finland. However, they differ substantially in their resistance to below 0°C temperatures in their natural hosts. T. nativa can live in frozen fox meat for years, whereas T. spiralis dies when frozen. In mouse muscle, the difference is not as evident; even T. nativa cannot maintain infectivity when kept at −20°C for 1 week. Crude larval protein extracts of these two parasite species were analyzed by two-dimensional gel electrophoresis (2DE). The protein patterns showed clear differences, but matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) peptide mass fingerprint followed by database searches failed to identify these proteins, suggesting that they may still be uncharacterized. The patterns compared after freezing treatment at −20°C revealed changes in the intensity of some protein spots. The antigenic differences of the species were analyzed with two-dimensional Western blots, which showed T. spiralis-specific proteins.     

Analysis of Yaba tumor poxvirus-induced proteins in infected cells by two-dimensional gel electrophoresis

Lack of inhibition of host protein synthesis by Yaba tumor poxvirus and comigration of viral and host proteins did not interfere with high resolution of virus-induced proteins when two-dimensional gel electrophoresis was employed. A total of 84 virus-induced proteins, 44 structural and 40 nonstructural, comprising 62.9% of the coding capacity of Yaba virus DNA, were detected. These proteins may be further categorized in two classes: early, synthesized in the presence of DNA inhibitor, Ara-c; and late, beginning at 6 hr p.i. with the bulk of viral protein synthesis occurring later than 30 hr p.i. The rate of synthesis of viral proteins fell into two patterns: group A, in which the rate of synthesis decreased; and, group B, in which the rate of synthesis increased after the initial appearance of the protein.     

Human polymorphic APO-LDL investigated by high-resolution two-dimensional electrophoresis

The APO part of a human polymorphic low density lipoprotein (LDL) was investigated by two-dimensional electrophoresis. Since these allotypic LDL were purified from sera by specific immunochromatography, the data presented here refer to only one molecular species of LDL. The two-dimensional electrophoresis revealed the presence of isoforms which were correlated to the serological phenotype. A hypothesis about APO-LDL gene organization is outlined.     

Interpretation of cerebrospinal fluid proteins by gel electrophoresis.

The use of polyacrylamide gel electrophoresis (PAGE) for the separation of proteins in cerebrospinal fluid (CSF) results in greater definition than does a "routine" method such as cellulose acetate electrophoresis. Unconcentrated CSF is easily separated into as many as 18 bands by the use of PAGE. By means of a modified PAGE method described in this paper, unconcentrated and untreated CSF is quickly and conveniently analyzed for protein constituents. This modification involves a continuous buffer environment, a pore-size concentration gradient and CSF in amounts of 0.1 to 0.4 ml. Sucrose addition is not necessary in this procedure. Whereas most central nervous system (CNS) disease states do not yield consistently distinctive protein patterns, some diseases, such as vascular disease, infectious meningitis and some metastatic tumors, yield significantly altered patterns. It is suggested that the chief value of CSF protein electrophoresis at the present time is to follow the course of a CNS disease.     

Analysis of immune complexes by two-dimensional gel electrophoresis

High resolution two-dimensional (2D) gel electrophoresis is useful for analysis of constituents of immune complexes (IC) in serum, provided that the samples for the analysis are prepared by a standardized and effective purification protocol. The details of the protocol, which involve gel permeation chromatography and adsorption with protein A-Sepharose, were worked out with a model system of radiolabeled antigen bound to antibody. With this protocol one can attain an over 50% recovery of the antigen, in a protein preparation purified over 1000-fold with respect to starting amounts in an initial 0.5 ml serum. With silver staining of the 2D gel, the model antigen was detectable at levels of 100 ng initial input. The analysis of eight normal sera and eight sera of patients with systemic lupus erythematosus (SLE) showed no clearly demonstrable differences, suggesting that the latter sera did not contain homogeneous antigens exceeding 100 ng within IC. In addition to IgG, albumin was seen in all preparations, probably due to complexing with immunoglobulin. Trace amounts of other constituents of the samples appeared in some gels, and the presence of C3 related material was detectable only by Western blotting.     

Characterization and identification of early proteins in Chlamydia trachomatis serovar L2 by two-dimensional gel electrophoresis.

The synthesis of early proteins from Chlamydia trachomatis serovar L2 was analyzed by two-dimensional gel electrophoresis. By pulse-label experiments, the synthesis of seven proteins was observed at 2 to 8 h postinfection before the major outer membrane protein was detected at 8 to 10 h after infection. The early proteins were synthesized throughout the 30-h period investigated, but the synthesis of three proteins of 75, 62, and 45 kilodaltons decreased from 26 to 30 h postinfection. Pulse-chase analysis showed that the signals from the same three proteins declined 26 to 30 h after infection. Three of the early proteins were identified as the S1 ribosomal protein, the GroEL-like protein, and DnaK-like protein, respectively.     

Identification of proteins from colorectal cancer tissue by two-dimensional gel electrophoresis and SELDI mass spectrometry

We investigated protein profiles obtained from colorectal tumor tissue and adjacent normal mucosa to identify tumor specific changes. Protein extracts of biopsis were separated by two-dimensional gel electrophoresis and >40 low-molecular mass proteins were identified by peptide fingerprinting using surface-enhanced laser desoption/ionization mass spectrometry (SELDI-MS). Among these, PACAP protein, hnrnp A1, flavin reductase, calgizzarin, NDK B (NM23-H2), cyclophilin A and smooth muscle protein 22-alpha showed significantly differential abundancy in the analyzed specimens. In addition, immunohistochemical analysis of tissue distribution and subcellular localization of some of the differentially expressed proteins demonstrated alterations in subcellular protein distribution. Further investigations are in progress to assess whether these differentially expressed proteins are associated with tumor development and tumor progression.     

Phenotypic characterization ofTheileria parva schizonts by two-dimensional gel electrophoresis

Biosynthetically radiolabelledTheileria parva schizonts were purified from bovine lymphoblastoid cells and their proteins were analyzed by two-dimensional gel electrophoresis and autoradiography. The protein spot patterns of schizont proteins from three stocks ofT. parva parva indicated that the phenotypic diversity among the stocks was minimal, with the Mariakani and Uganda stocks being identical and the Muguga stock showing only a few differences in minor spots. Comparison of the spot patterns of schizonts of threeT. parva subspecies showed thatT. p. parva andT. p. bovis differed in only one protein and thus could not be reliably distinguished on the basis of their protein differences. However,T. p. lawrencei showed several protein differences and could be distinguished easily from the other subspecies. Differences in schizont-protein spot patterns were also seen when two different cell lines were infected with the sameTheileria stabilate, when one cell line was infected with two different stabilates of the same stock and when uncloned and cloned infected cell lines were used. These results suggest the possibility that selection of phenotypically different parasites could occur in vivo or in vitro.II RAD publication 641     

A case of type III hyperlipoproteinaemia studied by acrylamide gel gradient electrophoresis.

A case of type III hyperlipoproteinaemia has been investigated before and after treatment, and the unusual serum lipoprotein patterns obtained by molecular exclusion or pore limit electrophoresis on acrylamide gradients have been compared.     

Precision and reliability of paraprotein determinations by high-resolution agarose gel electrophoresis

Agarose gel electrophoresis has recently replaced cellulose acetate electrophoresis as the preferred technique for monitoring paraprotein levels in patients with plasma cell dyscrasias. The authors studied the accuracy and precision of this method for paraprotein determination. Twenty-seven serum samples with paraprotein concentrations ranging from 5 to 73 g/L were aliquotted and assayed on 20 separate occasions, and the mean and standard deviation for the paraprotein concentration in each serum was established. Linear regression analysis showed that the standard deviation of paraprotein concentration (SD) increased as a function of paraprotein concentration (PC). For IgG paraproteins, the regression equation was SD = 0.041 (PC) + 1.06; R = 0.942; standard error = 0.32. For non-IgG paraproteins the equation was SD = 0.101 (PC) - 0.04; R = 0.851; standard error = 0.5. The accuracy of paraprotein determinations by the agarose gel electrophoretic technique was assessed by comparison with values obtained with the use of a previously validated enzyme-linked immunosorbent assay (ELISA) method for quantitation of IgG subclasses. Results obtained by the two methods were similar and highly correlated: (concentration by electrophoresis) = 0.921 (concentration by ELISA) + 0.46; R = 0.988; standard error = 0.34. The laser densitometric scanning procedure showed a loss of linearity above 60 g/L, indicating the need to dilute sera with very high paraprotein concentrations in order to obtain accurate results. A table is presented that should help pathologists who interpret such scans to determine whether small changes in paraprotein measurements occurring over time represent true changes in paraprotein concentration or merely reflect the analytic variability inherent in the technique.     

双向凝胶电泳脑脊液蛋白质提取方法的比较

 目的 对 3 种常用的双向凝胶电泳脑脊液蛋白质提取方法进行比较，优选脑脊液蛋白质提取方法。 方法 收集新诊断尚未经药物治疗的 16 例帕金森病患者和 7 例头痛患者的脑脊液，分别以 3 种不同的方法提取脑脊液中的蛋白质。①透析法：用 PluseOne 微透析试剂盒处理脑脊液样品。②丙酮沉淀法：以冷丙酮溶液处理脑脊液样品，丙酮的终浓度为 80%。③三氯醋酸（TCA）/丙酮沉淀法：以 4 倍体积的 10% TCA/丙酮溶液处理脑脊液样品，TCA 的终浓度为 8%，丙酮的终浓度为 80%。取沉淀物进行双向凝胶电泳，用银染法对电泳后的凝胶进行染色，比较经 3 种不同方法提取的脑脊液蛋白质的双向凝胶电泳图谱。 结果 经透析法处理样品的电泳图谱显示的蛋白点较清晰、较圆，条纹较少，但所见几乎都是高丰度蛋白，低丰度蛋白很难显现；经丙酮沉淀法处理样品的电泳图谱显示横竖条纹均较多，大部分蛋白堆积在凝胶的上部，下面的点也显示不清晰，蛋白点出现横向漂移；经 TCA/丙酮沉淀法处理样品的电泳图谱显示横条纹相对较少，低丰度蛋白能较清晰显现，样品中所含高丰度蛋白明显少于以上两种方法处理的样品。 结论 以 TCA/丙酮沉淀法提取蛋白质进行双向凝胶电泳时聚焦效果较好，而且同时去除了大部分白蛋白，使低丰度蛋白能很好地显现出来，优于丙酮沉淀法和透析法。     

Direct blotting with viable cells of protein mixtures separated by two-dimensional gel electrophoresis

A procedure is described which combines the high resolution power of two-dimensional (2D) gel electrophoresis with the advantage of direct probing with viable cells. This device permits the transfer by electroelution of 480 distinct fractions from a 2D gel into soluble phase. Transferred fractions are virtually nontoxic, thus allowing direct probing with viable cells. Using this procedure it was shown that T cells from normal healthy individuals recognized a multitude of Mycobacterium tuberculosis antigens and that the fine antigen recognition pattern of T cells changed after short-term culture in vitro. The application of this procedure to the verification of antigen purity at the T cell level and to the identification of antigens within crude bacterial lysates which are recognized by cloned T cells is described. This approach should be applicable to the rapid identification and characterization of any interesting T cell antigen, for example from important pathogens against which a subunit vaccine is desirable. Moreover, it could be helpful for the analysis of interactions between soluble ligands and their target cells.