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.     

Analysis, by two-dimensional gel electrophoresis, of ribosome crystal proteins

A ribosome crystal is an aggregate of ribosomes which are packed in a regular array. Preliminary experiments analysing the proteins from ribosome crystals by two-dimensional gel electrophoresis show that, although most proteins appear similar to those from polyribosomes, four extra proteins also seem to be characteristic of ribosome crystals.     

Matrix-assisted laser desorption mass spectrometric peptide mapping of proteins separated by two-dimensional gel electrophoresis: determination of phosphorylation in synapsin I.

A technique is described for the rapid, sensitive analysis of posttranslational modifications of proteins that have been separated by 2-dimensional electrophoresis and blotted onto a membrane with a cationic surface. The isolated protein spots visualized by reverse staining of the blotting membrane are excised, washed, and subjected to chemical (cyanogen bromide) and/or enzymatic (endoproteinase Lys-C) degradation directly on the membrane. The resulting mixture of peptide fragments is extracted from the membrane into a solution that is compatible with matrix-assisted laser desorption mass spectrometric analysis and analyzed without fractionation. Relatively accurate (+/- 1 Da) mass determination of these peptide fragments provides a facile and sensitive means for detecting the presence of modifications and for correlating such modifications with the differential mobility of different isoforms of a given protein during 2-dimensional electrophoresis. The technique is applied to the determination of sites of phosphorylation in synapsins Ia and Ib, neuronal phosphoproteins that are believed to function in the regulation of neurotransmitter release and are substrates for cAMP and Ca2+/calmodulin-dependent protein kinases, which appear to control their biological activity.     

Analysis of the Shewanella oneidensis proteome by two-dimensional gel electrophoresis under nondenaturing conditions

Proteomes are dynamic, i.e., the protein components of living cells change in response to various stimuli. Protein changes can involve shifts in the abundance of protein components, in the interactions of protein components, and in the activity of protein components. Two-dimensional gel electrophoresis (2-DE) coupled with peptide mass spectrometry is useful for the analysis of relative protein abundance, but the denaturing conditions of classical 2-DE do not allow analysis of protein interactions or protein function. We have developed a nondenaturing 2-DE method that allows analysis of protein interactions and protein functions, as demonstrated in our analysis of the cytosol and crude membrane fractions of the facultative anaerobe Shewanella oneidensis MR-1. Our experiments demonstrate that enzymatic activity is retained under the sample and protein separation methods described, as shown by positive malate dehydrogenase activity results. We have also found protein interactions within both the soluble and membrane fractions. The method described will be useful for the characterization of the functional proteomes of microbial systems.     

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.     

Rapid two-dimensional analysis of proteins by ultra-thin layer gel electrophoresis

Identification of qualitative and/or quantitative protein expression differences as well as characterization of specific cell proteomes would further advance molecular cell biology research. Today, one of the most commonly used tools for proteome analysis is two-dimensional gel electrophoresis. Although this technology is informative, it is extremely cumbersome, time-consuming and lacks automation and proper reproducibility. In this paper, we propose an automated separation/detection system capable of rapid two-dimensional analysis of proteins by ultra-thin layer gel electrophoresis with real time imaging of the separated components, using fiber optics based laser induced fluorescence technology. The approach is based on electric field mediated separation in capillary dimensions, along with noncovalent, "in migratio" fluorescent staining methodology. The advantage of the technology discussed over existing techniques is its simplicity, speed and good detection sensitivity.     

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.     

Preparation of membrane proteins for analysis by two-dimensional gel electrophoresis

In order to separate hydrophobic membrane proteins, we have developed a novel two-dimensional electrophoresis system. For the iso-electric focusing, agarose was used as a supporting matrix and n-dodecyl-beta-D-maltopyranoside was used as a surfactant. In combination with a previously developed Tris/MES electrophoresis system in the second dimension, distinct spots were reproducibly detected from hydrophobic membrane proteins whose grand average hydropathicity (GRAVY) exceed 0.3. In contrast to the immobilized pH gradient system, c-type heme was also visualized in this system.     

Analysis of normal and neoplastic lymphocyte surface-labeled proteins by two-dimensional polyacrylamide gel electrophoresis

Normal and neoplastic murine and human lymphocytes were surface-labeled by lacto-peroxidase-catalyzed radioiodination, and the cell lysates were subjected to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analyses, combining isoelectric focusing in the first dimension and sodium dodecyl sulfate-PAGE (SDS-PAGE) in the second dimension. 2D-PAGE autoradiogram patterns were reproducible and reflected differences in cell types. A string of spots with a Mr of 100K was tentatively identified as a new T-cell marker (Tp100) which was present in all murine and human T cells examined including human T lymphomas. Murine and human B cells displayed markers characteristic to B cells of each species with some similarities between them. Human lymphomas and murine cell lines showed markers which were absent or only weakly visible in normal cells. Thus, 2D-PAGE analysis of lymphocyte surface proteins proved to be a method useful for searching for various markers.     

Analysis of lymphocyte proteins from New Zealand black mice by two-dimensional gel electrophoresis

Splenic lymphocyte proteins from New Zealand Black (NZB) mice, which spontaneously develop autoimmune disease, and several control strains were analyzed by two-dimensional polyacrylamide gel electrophoresis. A number of strain- and age-related differences were observed, among which was the persistently elevated synthesis of two peptides, 12.5 KD and 10.5 KD, by spleen cells from older NZB mice. Although synthesis of these peptides was moderately high in young NZB and control mice, it diminished with age in control mice. These proteins were found in the cytoplasm and were not expressed on the plasma membrane nor secreted into the medium. Production of these proteins was restricted to B and null cells; T cells did not synthesize these peptides. These proteins appear to be indicators of disease activity, because their increased synthesis was associated with lymphocyte subset alterations associated with the onset of overt autoimmune disease in NZB mice.     

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 by two-dimensional polyacrylamide gel electrophoresis of the in vivo phosphorylation of ribosomal proteins derived from free and membrane-bound polysomes

Analysis of in vivo phosphorylation of mouse liver ribosomal proteins was performed by two-dimensional polyacrylamide gel electrophoresis following 32P-injection. Our method is special and differs from other eukaryotic systems reported in that all proteins separated on the first dimension gel are completely solubilized, moving quantitatively to the second dimension gel. Only ribosomes from polysomes were used, ensuring analysis of ribosomes actively engaged in protein synthesis. We resolved sixty-five distinct proteins from ribosomes from membrane bound or free polysomes. In both cases radioautography revealed similar labeled patterns with one highly phosphorylated ribosomal protein and five marginally labeled spots.     

Detection of tyrosine phosphorylated proteins by combination of immunoaffinity enrichment, two-dimensional difference gel electrophoresis and fluorescent Western blotting

We describe fluorescence-based 2-D gel electrophoresis methods for visualization of low abundant, cancer relevant tyrosine phosphorylated (pTyr) proteins. The methods investigated were fluorescent Western blotting and two-dimensional difference gel electrophoresis (2-D DIGE) for detection of non-enriched and immunoaffinity enriched pTyr protein patterns. The same anti-phosphotyrosine specific antibody, 4G10, was used for both approaches. The results from fluorescent Western blotting of total proteins and from enriched CyDye DIGE pre-labeled pTyr proteins showed similar down regulation of phosphorylation upon treating of cells from a cancer model system (K562 chronic myeloid leukemia cells) with imatinib. This treatment introduced a known perturbation of phosphorylation that enabled testing of these new approaches to analyze variations in tyrosine phosphorylation levels. Enrichment of pTyr proteins was found highly advantageous for the outcome. Out of a simplified 2-D DIGE experiment of immunoaffinity enriched control and treated pTyr proteins, differential analysis as well as protein identification by mass spectrometry (MS) was possible.     

Micro-scale two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins

Summary A specially designed apparatus and conditions are described for the rapid analysis of ribosomal proteins by two-dimensional gel electrophoresis on a micro scale. The resolution of proteins in electropherograms is comparable to that obtained with other systems, but because of miniaturization, only 0.5 to 1 g of each protein is required, and the entire procedure, including electrophoresis in both dimensions, and staining and destaining can be completed in 6 to 7 hours.     

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.     

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.     

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.     

Comparison of plant cytoplasmic ribosomal proteins by two-dimensional polyacrylamide gel electrophoresis

The electrophoretic mobilities of the cytoplasmic ribosomal proteins of several species of plants were compared using two-dimensional electrophoresis. The total number of proteins as well as the number of acidic and basic proteins in individual species varied markedly. Of the species examined, Triticum aestivum had the highest number of basic cytoplasmic ribosomal proteins and Hordeum vulgare had less than half as many. However, marked similarities were noted in the electrophoretic mobilities of many of the proteins, especially for wheat, rye, and barley and for peas and beans. There was a statistically significant positive correlation between the numbers of basic proteins in the species and their chromosome number.