Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis

A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P-40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a greater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when membrane proteins were extracted with phenol and precipitated with ammonium acetate in methanol; background staining and streaking were diminished and proteins were clearly resolved. This method makes possible the resolution required for meaningful qualitative and quantitative comparisons of protein patterns on two-dimensional gels of plant membrane proteins.     

Solubilization of membrane proteins for two-dimensional gel electrophoresis: identification of sarcoplasmic reticulum membrane proteins

Solubilization of membrane proteins for two-dimensional electrophoresis (2DE) is very difficult. In this study, we report the use of 1,2-diheptanoyl-sn-glycero-3-phosphatdiyl choline (DHPC) as a detergent to solubilize integral membrane proteins for 2DE. Rat ventricular microsomal fractions enriched with sarco(endo)plasmic reticulum (SR) membrane proteins were used as a model system. Compatibility of DHPC with a high concentration of urea increases the solubility of proteins compared with sulphobetaines or ASB-14. Peptide mass analysis assisted in the identification of key SR membrane proteins including SR Ca(2+) ATPase and other membrane proteins, which have not previously been reported on 2DE. These results suggest that DHPC is a better detergent for solubilizing membrane proteins and may be useful in generating proteomic maps for most complex organelles including SR.     

Solubilization, gel filtration and sedimentation behaviour of prolactin receptors from human ovarian tissue

Receptors for 125I-labelled human prolactin have been identified in the crude membrane fraction isolated from human ovarian tissue. The non-ionic detergent Triton X-100, has been used to solubilize the membrane fraction. The presence of the receptor in the detergent extract was demonstrated by gel filtration and sucrose density gradient centrifugation. The binding was time-temperature dependent, being maximal at 23 degrees C after 15 h of incubation. Large amounts of other peptide hormones did not inhibit the binding of 125I-labelled human prolactin. The binding Scatchard analysis demonstrated that the affinity of the soluble receptor (Ka 1.13 +/- 0.15 X 10(10) M-1) for the labelled hormone was slightly greater than that of the crude membrane fraction (Ka 0.91 +/- 0.12 X 10(10) M-1). The binding capacity of the solubilized receptor was also significantly greater than that seen in the particulate before solubilization. The apparent Stokes radius of the solubilized receptor was estimated to be 57 A and that the hormone-receptor complex 60 A. The sedimentation coefficient of the solubilized receptor was 7.0 +/- 0.1 s, whereas that of the hormone-receptor complex was 7.5 +/- 0.2 s.     

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.     

Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis

A problem in proteomic analysis of lung cancer tissue is the presence of complex components of different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma, and adenocarcinoma). The efficient solubilization of protein components before two-dimensional electrophoresis (2-DE) is a very critical. Poor solubilization has been associated with a failure to detect proteins and diffuse, streaked and/or trailing protein spots. Here, we have optimized the solubilization of human lung cancer tissue to increase protein resolution. Isoelectric focusing (IEF) rehydration buffer containing a thiourea–urea mixture provided superior resolution, whereas a buffer without thiourea yielded consistently poor results. In addition, IEF rehydration buffers containing CHAPS and DTT gave superior resolution, whereas buffers containing Nonidet P-40 (NP-40) and/or Triton X-100 did not. A tributylphosphine-containing buffer gave consistently poor results. Using optimized conditions, we used 2-D gel analysis of human lung cancer tissue to identify 11 differentially-expressed protein spots by MALDI-mass spectrometry. This study provides a methodological tool to study the complex mammalian proteomes.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis

Summary We describe a genetic polymorphism of cytosol polypeptide with mol. wt. of 38,000 detected in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes by two-dimensional gel electrophoresis. Three different electrophoretic phenotypes (type 1-1, 2-1, 2-2) of the polypeptide have been identified in a Japanese population. Family and population studies indicate that three phenotypes are determined by two common alleles at a single autosomal locus. Since the polypeptide is mainly present in cytosol of cells, we propose that the polypeptide be temporarily designated as cytosol polypeptide with mol. wt. of 38,000 (CP 38) and that the gene for CP 38 be designated as CP 38. The gene frequencies of two common alleles (CP 38 ¹ and CP 38 ²) are 0.899 and 0.101, respectively, in a Japanese population. The data on gel filtration of cytosol proteins on a Sephadex G-100 column suggest that CP 38 exists as a dimer in the cytosol. CP 38 was observed in the wide range of different cells, including B-lymphoblastoid cells, adult skin fibroblasts, HeLa cells, and erythrocytes. In 11 out of 72 individuals, the phenotypes of CP 38 were different from those of adenosine deaminase which is similar to CP 38 in subunit size, cell distribution, and allele frequencies. These data indicate that CP 38 is a new polymorphic polypeptide encoded by an autosomal locus.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis

Summary We describe a genetic polymorphism of cytosol polypeptide with mol.wt. of 20,000 detected in lymphocytes the arythrocytes by two-dimensional gel electrophoresis. Three different electrophoretic phenotypes (type 1-1, 2-1, and 2-2) of the polypeptide have been identified in a Japanese population. Family studies indicate that the phenotypes are determined by two common alleles at a single autosomal locus. The polypeptide is present in the cytosol of various kinds of cells and is abundant in erythrocytes. The data on a gel filtration of the erythrocyte cytosol proteins on a Sephadex G-100 column suggest that the polypeptide exists as a dimer in cells. In nine out of 79 individuals, the phenotypes of the polypeptide were different from those of glyoxalase 1 (GLO1) which has similar properties in subunit size, cell distribution, and allele frequencies. These date indicate that the polypeptide with mol. wt. of 20,000 is a new polymorphic cellular polypeptide. We propose that the polypeptide be temporarily designated as cytosol polypeptide with mol. wt. of 20,000 (CP20) and that the gene for CP20 be designated as CP20. The gene frequencies of two common alleles (CP20 ¹ and CP20 ²) are 0.955 and 0.045, respectively, in a Japanese population.     

Characterization of N-ethylmaleimide-reactive proteins from human tonsillar ribosomes by two-dimensional polyacrylamide gel electrophoresis.

Human tonsillar 80-S ribosomes were 17% and 43% inactivated by 1 mM N-ethylmaleimide after 12 min at 30 or 37 degrees C, respectively. The ribosomes were unaffected by the reagent during the same period of time at 0 or 20 degrees C. 4, 12, 27 and 59 sulfhydryl groups per 80-S ribosomes were found labeled by 1 mM N-ethyl[14C] maleimide after 12 min at 0, 20, 30 or 37 degrees C, respectively. The analysis of radioactively labeled proteins by two-dimensional gel electrophoresis revealed the following: after 3 min at 37 degrees C only two 40-S proteins, S3 and S7, displayed a significant amount of label. After 12 min at 37 degrees C, there was a several-fold increase in the extent of radioactivity found in each of these proteins and, additionally, S1, S2, S4, S5, S15, S22 and S31 were also found among labeled 40-S proteins. S3 appeared to be the most N-ethylmaleimide-reactive 40S protein. After 3 min at 37 degrees C, L10, L17, L20 (and/or S20), L26, L32 and L33, and after 12 min at 37 degrees C, additionally L1, L2, L7, L9, L11, L15, L16, L18, and L25 were labeled among 60-S proteins. l17 and 32 were the most N-ethylmaleimide-reactive proteins under these conditions. After 12 min at 37 degrees C, approx. 26% and 39% of the radioactivity incorporated into the 80 S or 60 S ribosomal protein, respectively, was found in these two proteins. After 12 min at 0 degrees C, S3, L17, L32 and L33 were the only labeled proteins.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis:

Summary Approximately 250 phytohemagglutinin (PHA)-stimulated peripheral blood lymphocyte polypeptides from three unrelated healthy males were compared by high-resolution two-dimensional gel electrophoresis and double-label autoradiography. Comparisons by all possible pairwise combinations of [¹⁴C]leucine-labeled proteins from an individual and [³H]leucine-labeled proteins from another revealed that only three polypeptides differed qualitatively among the three individuals. The degree of variation in lymphocyte polypeptides between different individuals was similar to that in fibroblast polypeptides reported previously. Among the three variant polypeptides, two polypeptides with mol.wt. 64,000 and mol. wt. 37,000 coexisted with a polypeptide with the same molecular weight, and they showed the behavior expected of two allelic gene products separated in the isoelectric focusing dimension by charge differences. Analysis of [¹⁴C]leucine labeled peripheral blood lymphocyte proteints, from the parents of each individual, by two-dimensional gel electrophoresis indicated that the variant polypeptides with mol. wt. 64,000 and mol. wt. 37,000 in the propositus were inherited from one of his parents. The data indicate that genetic analysis of PHA-stimulated peripheral blood lymphocyte proteins is feasible by high-resolution two-dimensional gel electrophoresis in combination with double-label autoradiography and pedigree analysis.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis:

Summary Three different electrophoretic types (1-1, 2-1 and 2-2) of a human cellular polypeptide with molecular weight of 31000 have been identified by the analysis of PHA-stimulated peripheral blood lymphocyte proteins using high resolution two-dimensional gel electrophoresis. Family and population studies indicate that the three phenotypes of the polypeptide are determined by two common alleles at a single autosomal locus. The 31k polypeptide appears to be present as a monomer in the cytosol in a wide range of different cell types, including permanent lymphoblastoid cell lines, fibroblasts and HeLa cells. In an individual with the 31k polypeptide type 2-2, the phenotypes of adenosine deaminase and uridine monophosphate kinase were both type 1. These data indicate that the 31K polypeptide is a new polymorphic protein encoded by a new autosomal locus. It is proposed that the polypeptide and its locus be temporarily designated cytosol 31k polypeptide (C31k polypeptide) and C31P, respectively. In a Japanese population, the gene frequencies of C31P ¹ and C31P ² were 0.940 and 0.060, respectively. The C31k polypeptide type 2-2 appears to be a molecular weight variant as well as a charge variant.     

High resolution two-dimensional gel electrophoresis of human erythrocyte membrane proteins.

Three different two-dimensional (2-D) gel electrophoretic techniques have been modified to provide high resolution of human erythrocyte membrane proteins. The resulting gels were referenced to the established one-dimensional (1-D) sodium dodecylsulfate (SDS) gel electrophoretic profile, and the effects of endogenous proteolysis and cytosolic contamination were studied. It is concluded that in vitro proteolysis and cytosolic contamination do not contribute significantly to the patterns observed on the 2-D gels, under the conditions used for erythrocyte ghost preparation. The procedures require only small quantities of blood; as many as twenty 2-D gel profiles can be obtained from 5 ml of blood. The combination of nonequilibrium isoelectric focusing (IEF) in the first dimension, SDS electrophoresis in the second dimension, and very sensitive silver staining techniques resolves more than 250 individual protein spots. This appears to be the most useful single procedure for the analysis of red cell membrane proteins. Membrane protein profiles from patients with Duchenne muscular dystrophy, Wernicke-Korsakoff syndrome, and acanthocytosis with degeneration of the basal ganglia were compared with normal controls. The patterns for Duchenne muscular dystrophy and Wernicke-Korsakoff syndrome were not different from normal patterns. The pattern for the patient with acanthocytosis and degeneration of the basal ganglia consistently showed a high level for one protein in the 100,000 mol. wt. range.     

Comparison of membrane proteins from benign and malignant human thyroid tissues by two-dimensional polyacrylamide gel electrophoresis

In this study two-dimensional (2D) polyacrylamide gel electrophoresis with silver staining was used to analyze cellular membranous proteins of various normal and pathological human thyroid tissues. The aim was to understand the differences in cellular membranous proteins between these tissues, which would aid in the differential diagnosis of thyroid malignancy. Characteristic protein spots had a molecular mass of 50–64 kDa and a pI of 5.7-6.5. There were two groups of isoform protein spots in this area. The higher-molecular-mass group was found in follicular thyroid cancer tissues which and was not visible in normal thyroid tissues. The low-molecular-mass group was found in follicular carcinoma or adenoma tissues and was detected in one to three spots. The papillary thyroid carcinoma tissues gave different 2D gel maps. There were few spots of papillary thyroid carcinoma tissue membranous proteins within the examined area. The 2D gel maps may be used for differential diagnosis of follicular neoplasm. The characteristics of these protein spots require further investigation.     

Developmental appearance of proteins identified by two-dimensional gel electrophoresis in mouse gonadal tissue

Gonadal protein patterns of the mouse were studied during fetal development by two-dimensional gel electrophoresis. Fetal mice at days 8.5, 10.5, 12.5, and 14.5 post-coitum were analyzed for male or female specific proteins. Although no sex specific proteins were found, several proteins were found which were expressed in significantly different amounts in the two sexes at about the time of gonadal differentiation. Hence, quantitative differences, rather than qualitative ones, could be related to the initiation of testis or ovary development.     

Microsequence analysis of winged bean seed proteins electroblotted from two-dimensional gel

Electroblotting method employing a semidry blotting apparatus for the subsequent protein microsequence analysis (Hirano, 1987) was improved. This method is convenient and allows rapid and efficient transfer of the proteins from a polyacrylamide gel (1 mm thick) onto the Polybrene-coated glass-fiber sheet or polyvinylidene difluoride membrane filter in only 20 min. The electroblotted proteins could be sequenced directly with the gas-phase protein sequencer at a 20-pmole level. This method was applied to the sequence analysis of winged bean seed proteins. A portion of the crude extracts from only one-twentieth of a seed of the winged bean was separated by two-dimensional polyacrylamide gel electrophoresis and electroblotted, and the N-terminal amino acid sequences of the blotted proteins were analyzed. The sequences of about 60% of the blotted major proteins, including nine Kunitz trypsin inhibitor-like proteins with heterogeneity in the N-terminal sequences, a protein that has a homologous sequence to the leghaemoglobin, nitrogen-fixing root nodule-specific protein, and a soybean basic 7S globulin-like protein could be easily identified.     

Separation of histones from contaminating ribosomal proteins by two-dimensional gel electrophoresis

Sea urchin histones can be separated from ribosomal proteins by two-dimensional gel electrophoresis. Electrophoresis on Triton X-100/6 m urea gels in the first dimension results in preferential retardation of the histones, which then migrate more rapidly than ribosomal contaminants on SDS gel electrophoresis in the second dimension. The advantages and generality of the system are discussed.     

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.     

Triple-spot proteins in two-dimensional gel electrophoresis.

A triple-spot pattern of polypeptides occurring in two-dimensional gel electrophoresis of proteins is described. The presence of a mutant protein, Pc 1 Duarte, which results in a splitting of all three polypeptides, is evidence that they are produced by the same gene. This pattern is seen in about 1% of the proteins from a variety of sources. Typically, about 50% of the protein occurs as a single major spot, the remainder occurring as two polypeptides with an additional negative charge and slightly different molecular weight. The reproducibility of this pattern implies a functional significance which is presently unknown. The implication of this configuration for patterns seen by one-dimensional gel electrophoresis is discussed.     

Study on proteins from yeast cytoplasmic ribosomes by two-dimensional gel electrophoresis

Summary Proteins of yeast cytoplasmic ribosomes were analyzed by two different methods of two-dimensional gel electrophoresis: run at pH 8.6 in 1-D¹ and at pH 4.6 in 2-D (Method A); run at pH 5.0 in 1-D and in the presence of sodium dodecyl sulfate in 2-D (Method B). The numbers of proteins estimated were 28 (Method A) and 29 or 30 (Method B) in the 40S small subunit, and 40 (Method A) and 41 (Method B) in the 60S large subunit, respectively. Molecular weights of proteins in the small and the large subunits were found to be less than 40,000 and 60,000 respectively.