Optimizing protein solubility for two-dimensional gel electrophoresis analysis of human myocardium

In order to maximize the myocardial proteome observed by two-dimensional gel electrophoresis (2-DE), the effect of (1) either an ionic or different zwitterionic detergents during tissue homogenization and (2) altering the "standard" detergent for isoelectric focusing (3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS)) to either the zwitterionic detergent amidosulfobetaine-14 (ASB-14) or N-decyl-N-N'-dimethyl-3-ammonio-1-propane sulfonate (SB3-10) was investigated. Sodium dodecyl sulfate was shown to be a superior detergent for extraction of proteins during homogenization of cardiac tissue compared to the detergents ASB-14, SB3-10 or CHAPS. Additionally, both ASB-14 and SB3-10 exhibited better extraction than CHAPS for distinct regions of two-dimensional gels. In most cases, the best combination of homogenization and focusing conditions did not involve the use of the same detergent. Specifically, it was found that the ability to mix homogenization and focusing conditions can allow one to obtain an optimum balance between the resolution and number of protein spots obtained in 2-DE analysis of cardiac tissue. An excellent initial combination of buffers to utilize for the general examination of cardiac proteins was determined to be initial homogenization in a buffer containing ASB-14 followed by focusing in a buffer containing CHAPS.     

Identification of human hepatocellular carcinoma-related biomarkers by two-dimensional difference gel electrophoresis and mass spectrometry

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death throughout the world. Although hepatitis B or C viral infections are main risk factors for HCC, the molecular mechanisms leading to HCC formation have not been clarified. To reduce the mortality and improve the effectiveness of therapy, it is important to search for changes in tumor-specific biomarkers whose function may involve in disease progression and which may be useful as potential therapeutic targets. In this study, we employed two-dimensional difference gel electrophoresis (2D-DIGE) combined with nano flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) to investigate differentially expressed proteins in HCC. For each of eight HCC patients, Cy3-labeled proteins isolated from tumor tissue were combined with Cy5-labeled proteins isolated from the surrounding nontumor tissue and separated by 2D gel electrophoresis along with a Cy2-labeled mixture of all tumor and nontumor samples as an internal standard. Thirty-four protein spots corresponding to 30 different proteins were identified by nanoLC-MS/MS as showing significant change (paired t-test, p < 0.05) in the level of expression between tumor and nontumor tissues. Sixteen proteins were up-regulated and 14 were down-regulated in HCC; they seem to play important roles in a variety of pathways including glycolysis, fatty acid transport and trafficking, amino acid metabolism, iron and xenobiotic metabolism, ethanol metabolism, cell cycle regulation, cytoskeleton, and stress. A remarkable finding is the up-regulation of 14-3-3gamma protein in HCC. 14-3-3 isoforms had been linked to carcinogenesis because they are involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation, and differentiation. In conclusion, 2D-DIGE is an efficient strategy that enables us to identify differentially expressed proteins in HCC. Identification of potential biomarkers, such as the pinpointing of 14-3-3gamma in our findings, may provide further useful insights into the pathogenesis of HCC.     

Mapping proteins from various structural regions of human kidney by two-dimensional electrophoresis]

The protein composition of various structural divisions of human kidney was studied using two-dimensional electrophoresis. Two-dimensional electrophoregrams of the cortical substance of human kidney revealed 165 polypeptide fractions within the pH range of 4.5-7.5, having molecular masses of 10 to 330 kDa. Electrophoresis of glomerular proteins gave 155 fractions with M(r) = 15-300 kDa, whereas fractionation of glomerular basement membrane proteins gave 40 fractions with M(r) = 30-330 kDa within the same range of pH. The M(r) values for all fractions and the relative electrophoretic mobility in the forward direction were determined. A comparative analysis of the electrophoregrams was conducted. The data obtained were used to construct two-dimensional maps of the cortical substance and glomerular proteins of human kidney.     

Identification and mapping of human saphenous vein medial smooth muscle proteins by two-dimensional polyacrylamide gel electrophoresis

Changing smooth muscle phenotype and abnormal cell proliferation are important features of vascular pathology, including the failure of saphenous vein bypass grafts. We have characterised and mapped protein expression in human saphenous vein medial smooth muscle, using two-dimensional (2-D) polyacrylamide gel electrophoresis. The 2-D system comprised a nonlinear immobilised pH 3-10 gradient in the first dimension (separating proteins with isoelectric point values between pH 3-10), and 12%T total gel concentration sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension (separating proteins in the range 14,000-200,000 Daltons). Using a combination of peptide mass fingerprinting by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and partial amino acid sequencing by nanospray tandem mass spectrometry, a subset of 149 protein spots was analysed, with 129 protein spots being identified and mapped. The data presented here are an important addition to the limited knowledge of venous medial smooth muscle protein expression in vivo. Our protein map will facilitate the identification of proteins differentially expressed in human saphenous vein bypass grafts. In turn, this may lead to the elucidation of molecular events involved in saphenous vein bypass graft failure. The map should also provide a basis for comparative studies of protein expression in vascular smooth muscle of varying origins.     

Separation and identification of rice prolamins by two-dimensional gel electrophoresis and amino acid sequencing

There are difficulties in detecting and separating rice prolamin polypeptides by 2D-PAGE analysis because prolamin polypeptides are insoluble, and the amino acid sequences show high homology among them. In this study, we improved the prolamin extraction method and the 2D-PAGE procedure, and succeeded in separating prolamin polypeptide species by 2D-PAGE and in identifying major prolamin polypeptide sequences.     

Identification of protein carbonyls after two-dimensional electrophoresis

The oxidative modification of proteins plays a major role in a number of human diseases, but identity of the specific proteins that are most susceptible to oxidation has posed a difficult problem. Protein carbonyls are increased after oxidative stress, and after derivatization with 2,4-dinitrophenyl hydrazine (DNP) they can be detected by various analytical and immunological methods. Although high resolution two-dimensional electrophoresis (2-DE) can resolve virtually all proteins present in a cell or tissue it has been difficult to determine the oxidized proteins because the DNP-derivatization process alters the isoelectric points of proteins, and additional procedures must be utilized to remove reaction byproducts. These additional procedures can lead to loss of sample, and poor isoelectric resolution on immobilized pH gradient (IPG) strips. We have developed a method that allows the IPG strips to be derivatized with DNP directly following isoelectric focusing of the proteins. This method allows the visualization of oxidized proteins by 2-DE with high reproducibility.     

Combination of two-dimensional electrophoresis and shotgun peptide sequencing in comparative proteomics

Two-dimensional electrophoresis (2-DE) and shotgun peptide sequencing are the two major technologies to compare the expression profile of proteins, which is also referred to as comparative proteomics or quantitative proteomics. Although the methodologies, such as difference gel electrophoresis for 2-DE and isotope-coded affinity tags for shotgun peptide sequencing, have made rapid progress, these two approaches have their own strengths and weaknesses. Therefore, the combination of the two methodologies is beneficial for the purpose of better comparative proteomics, especially in comprehensive coverage of the proteome and protein information such as post-translational modifications.     

Identification of grass pollen allergens by two-dimensional gel electrophoresis and serological screening

Approximately 50% of allergic patients are sensitized against grass pollen allergens. The characterization of specific immunoglobulin E (IgE) reactivity to allergen components in pollen-allergic patients is fundamental for clinical diagnosis and for immunotherapy. Complex allergen extracts are commonly used in diagnostic tests as well as in immunotherapy preparations, but their composition in single allergenic molecules is only partially known. Diagnostic tests which utilize recombinant or immuno-purified allergens have been made available in clinical practice. They allow to obtain specific profiles of IgE reactivity, but the panel of available molecules is far from complete. Here, we used a proteomic approach in order to detect grass allergens from a natural protein extract. A five-grass pollen extract used for diagnosis and immunotherapy was resolved by two dimensional gel electrophoresis (2-DE), and assayed with 9 sera from pollen-allergic patients whose sensitization profile was dissected by using IgE reactivity to recombinant allergens. 2-DE immunoreactivity patterns were matched with IgE reactivity to identify protein spots as candidate allergens. Identity was confirmed by mass spectrometry analysis. We identified 6 out of 8 expected clinically relevant allergens in the natural grass extract. Moreover, we identified different molecular isoforms of single allergens, thus obtaining a more detailed profile of IgE reactivity. Some discrepancies in protein isoform profile and sera immunoreactivity between recombinant and native allergen 5 from Phleum pratense were observed and a new putative allergen was described. The proteomic approach applied to the analysis of a natural allergen allows the comprehensive evaluation of the sensitization profile of allergic patients and the identification of new allergens.     

Identification of phagocytosis-associated surface proteins of macrophages by two-dimensional gel electrophoresis

Two-dimensional PAGE (P. Z. O'Farrell, H. M. Goodman, and P. H. O'Farrell. 1977. Cell. 12:1133-1142) has been employed to assess the effects of antibody-dependent phagocytosis on the cell surface protein composition of RAW264 macrophages. Unilamellar phospholipid vesicles containing 1% dinitrophenyl-aminocaproyl-phosphatidylethanolamine (DNP- cap-PE) were used as the target particle. Macrophages were exposed to anti-DNP antibody alone, vesicles alone, or vesicles in the presence of antibody for 1 h at 37 degrees C. Cell surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination at 4 degrees C. After detergent solubilization, membrane proteins were analyzed by two- dimensional gel electrophoresis. The resulting pattern of spots was compared to that of standard proteins. We have identified several surface proteins, not apparently associated with the phagocytic process, which are present either in a multichain structure or in several discretely charged forms. After phagocytosis, we have observed the appearance of two proteins of 45 and 50 kdaltons in nonreducing gels. In addition, we have noted the disappearance of a 140-kdalton protein in gels run under reducing conditions. These alterations would not be detected in the conventional one-dimensional gel electrophoresis. This evidence shows that phagocytosis leads to a modification of cell surface protein composition. Our results support the concept of specific enrichment and depletion of membrane components during antibody-dependent phagocytosis.     

Identification of enzymes and activity from two-dimensional gel electrophoresis

Identification of proteins with enzymatic activity by mass spectrometry (MS) and concomitant determination of function by screening enzyme activity from two-dimensional gel electrophoresis (2DE) is one of the challenges of gel-based proteomics. In this protocol, proteins are extracted from spinal cord tissue followed by 2DE with in-gel digestion and identification by matrix-assisted laser desorption/ionization. Protein spots identified as possible enzyme of interest are punched, eluted by SDS-containing Tris buffer and renatured by buffers under reductive conditions. Enzyme activity is determined using micromethods. Within about 4 weeks, a structural and functional map can be generated and MS identification can be validated, complementing immunochemical methods. 2DE separation can be seen as a prepurification step and therefore allows activity assays from minute amounts of protein as provided in a 2DE gel spot; the method may be an alternative to the time-consuming use of recombinant enzyme techniques.     

Identification of the Salmonella typhimurium cysB gene product by two-dimensional protein electrophoresis.

Examination of two-dimensional electropherograms of proteins from wild-type Salmonella typhimurium and 16 different cysB strains permitted the identification of a single 34,500-dalton polypeptide chain with a pI of 7.6 that was the product of cysB. Exclusion chromatography indicated that the native cysB protein is a multimer of at least two and probably four or more such subunits.     

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.     

Analysis of human class I antigens by two-dimensional gel electrophoresis

Class I antigens were isolated by immunoprecipitation from cell extracts prepared from mitogenically stimulated and internally radiolabeled peripheral blood lymphocytes (PLBs). The precipitating antibodies used are monomorphic and recognize a determinant on the heavy chain of HLA-A, B, C antigens regardless of their allelic specificities when complexed with ₂m, or determinants on ₂m itself. Comparison of class I molecules isolated from 25 different homozygous typing cels (HTC) and analyzed by two-dimensional (2-D) gel electrophoresis allowed the identification of those HLA-A,13 locus specificities most common in the European Caucasoid population. Class I antigens isolated from HTC that are HLA identical are biochemically indistinguishable also. Evidence was obtained for the expression of additional class I antigens besides the HLA-A, B, C locus products: for some haplotypes, up to six class I genes may be active in mitogenically activated PBLs. No differences in molecular weight and isoelectric point of the class I heavy chains were observed between the antigens recognized by W6/32, the anti-heavy chain reagent, and anti- ₂m reagents. The nature of the mitogenic stimulus, i. e., pokeweed mitogen or phytohemagglutinin, was irrelevant with respect to the class I antigens isolated by this method. Using the HTCs as reference, a panel of HLA-B27 positive heterozygous cells was analyzed. Two types of HLA-B27 antigens, distinct by CML typing were represented. These two forms differed also in their biochemical properties. In addition, we obtained evidence for the existence of an A2 variant. This finding was likewise confirmed by CML typing.     

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.     

Analysis of the heterogeneity of human collagens by two-dimensional polyacrylamide-gel electrophoresis.

The heterogeneity of the CNBr-cleavage peptides of human types I, II, III and V collagens were studied by using two-dimensional electrophoresis combining non-equilibrium pH-gradient-gel electrophoresis and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Specific 'maps' were produced by the peptides obtained from the chains of each type of collagen, and most peptides had at least three charged forms of the same molecular weight. Specific 'maps' were also produced by the peptides of types I, III and V collagens from insoluble dermis and the peptides of types I and V collagens from decalcified bone. The alpha 1(I) CB7 and alpha 1(I) CB8 and the alpha 2 CB4 peptides obtained from the type I collagens of these tissues contained the same number of charged components, but there was a relative increase in the more basic components in bone. Some aspects of the involvement of the alpha 1(I) CB6 and the alpha 1(III) CB9 peptides in cross-linkages were also studied. The recovery of the alpha 1(I) CB6 peptide from bone and dermis was decreased and the alpha 1(III) CB9 peptide was not detected in dermis. Additional peptides, which were probably cross-linked peptides involving the alpha 1(I) CB6 peptide, were also observed.     

Identification of tissue proteins by amino acid analysis after purification by two-dimensional electrophoresis

Mouse brain proteins were separated by two-dimensional electrophoresis (2-DE). The proteins of a section of the 2-DE pattern were blotted onto hydrophobic membranes and 43 of them were excised and hydrolyzed by liquid-phase hydrolysis. The amino acid composition of these proteins was determined by orthophthaldialdehyde precolumn derivatization and compared with the compositions of known proteins stored in the NBRF sequence database. An identification program named ASA was developed for this purpose. The ASA program includes correction and weighting factors, data reduction by molecular weight windows, and exclusion or inclusion of certain organisms as desired. As a control, eight test proteins and five well-known proteins from mouse brain, all separated by 2-DE, were correctly identified by the program. Out of the 43 brain proteins selected, 19 were identified with high confidence.     

Identification of initiation factors and ribosome-associated phosphoproteins by two-dimensional polyacrylamide gel electrophoresis.

A two-dimensional polyacrylamide gel electrophoresis procedure has been used to identify initiation factors rapidly in the high-salt-wash fraction from reticulocyte ribosomes. Initiation factors are identified by relative mobility and by co-electrophoresis with purified factors. A creatine phosphate/ATP/GTP/Pi exchange system is described which has been used to maintain [gamma-32P]ATP and [gamma-32P]GTP at constant specific activity in the cell-free protein-synthesizing system. Phosphorylated proteins associated with the protein-synthesizing complex have been identified using a combination of the two procedures. The salt-wash fraction contains eight major phosphorylated proteins and a number of minor ones. Two phosphorylated proteins are observed to comigrate with two of the three subunits of eukaryotic initiation factor 2 (eIF-2), the initiation factor involved in binding Met-tRNAf onto the 40-S subunit and promoting dissociation of 80-S ribosomes. eIF-4B, one of the proteins involved in binding mRNA to 40-S subunits is also phosphorylated. The remainder of phosphorylated proteins in the high-salt-wash fraction are not previously characterized initiation factors and have not been identified further. Two of the six phosphoproteins associated with the salt-washed ribosomes comigrate with ribosomal proteins; one is the major phosphorylated protein in 40-S ribosomal subunits, the other is an acidic protein.     

Identification of surface proteins of Helicobacter pylori by selective biotinylation,affinity purification,and two-dimensional gel electrophoresis

Helicobacter pylori is a widespread human pathogen that can cause gastric ulcers and cancer. To identify surface proteins that may play a role in pathogen-host interactions and represent potential targets for the control of this infection, we selectively biotinylated intact H. pylori with the hydrophilic reagent sulfosuccinimidyl-6-(biotinamido)-hexanoate and purified the labeled proteins by membrane isolation, solubilization, and affinity chromatography. After separation of 82 biotinylated proteins on two-dimensional gels, 18 were identified with comparison to proteome data and peptide mass fingerprinting. Among the identified proteins, 9 have previously been shown to be surface-exposed, 7 are associated with virulence, and 11 are highly immunogenic in infected patients. In conclusion, this generally applicable combined proteome approach facilitates the rapid identification of promising targets for the control of H. pylori and might be applicable to numerous other human pathogens although larger biotinylation reagents might be required in some cases to prevent permeation of porin channels in the outer membrane.