Solubilization of cellular membrane proteins from Streptococcus mutans for two-dimensional gel electrophoresis

Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is due to low abundancy, poor solubility, and inherent hydrophobicity leading to self-aggregation during the first dimension. In this study, membrane proteins from the Gram-positive bacterium Streptococcus mutans were solubilized using three different methods and evaluated by 2-DE. In the first method, the extraction was performed using sodium dodecyl sulfate (SDS) followed by solubilization with a chaotropic buffer and precipitation with methanol/chloroform. The second method was based on temperature-dependent phase partitioning using Triton X-114 followed by purification using the ReadyPrep 2-D clean-up kit from Bio-Rad. The third method involved extraction using the organic solvents trifluoroethanol (TFE) and chloroform, which produced three separate phases. The upper aqueous phase, enriched with TFE, gave the highest overall protein yield and best 2-DE resolution. Protein spot identification by nanoelectrospray quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS) revealed known membrane and surface-associated proteins. This is the first report describing the successful solubilization and 2-D electrophoresis of membrane proteins from a Gram-positive bacterium.     

A two-dimensional electrophoresis map of Chinese hamster ovary cell proteins based on fluorescence staining

We report on the development of a detailed two-dimensional electrophoresis map of Chinese hamster ovary (CHO) cell proteins based on fluorescence staining and tandem time-of-flight (TOF/TOF)-mass spectrometry. We observed a 71% success rate in the identification of proteins even though the CHO genome is not sequenced. The map consists of 224 protein identifications present in 274 two-dimensional gel electrophoresis (2-DE) gel spots. We have also initiated a study of the phosphoproteome using a commercially available phosphoprotein-specific fluorescent stain. Using this stain, we observe 672 phosphorylated proteins, including many proteins known to be phosphorylated, which is 36% of the proteins we visualized with a total protein stain and consistent with expectations.     

Analysis of human tear proteins by two-dimensional electrophoresis.

Human tear proteins in the conjunctival sac were separated on the basis of the differences in their isoelectric points and molecular weights using micro two-dimensional electrophoresis combined with immunoblotting. The two-dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis were compared with those from normal individuals. We also measured integrated intensities of seven protein spots, lactoferrin (LF), albumin and five specific tear proteins (STP), to examine differences in the amounts of these proteins in tears from normal individuals of different sexes. In the tears from patients with conjunctivitis, secretory immunoglobulin A (IgA), LF and STP spots were stained more weakly, whereas the albumin spot was stained more strongly as compared with those from normal individuals. Furthermore, haptoglobin and IgG spots appeared in the tears from patients with conjunctivitis. These were more prominent in the tears from patients with severe conjunctivitis. There were significant differences in the amounts of LF and two kinds of STPs in the different sexes. The amounts of these proteins were larger in females.     

Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis

The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)-anchored proteins y two-dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent-resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI-anchored proteins. Although the GPI-anchored proteins were readily separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these proteins were undetectable on two-dimensional (2-D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent-resistant membranes such as caveolin-1 and flotillin-1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI-anchored proteins required cleavage of the lipid tail with phosphatidylinositol-specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI-anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI-anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N-glycosylation. Pretreatment with N-glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N-linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray-tandem mass spectrometry as illustrated for the GPI-anchored protein, Thy-1.     

Comparative analysis of brain proteins from p53-deficient mice by two-dimensional electrophoresis

p53 is a tumor suppressor protein that regulates many cellular processes including the cell cycle, DNA repair, and apoptosis. It also serves as a critical regulator of neuronal apoptosis in the central nervous system (CNS). To elucidate the role of p53 in the CNS, brain proteins of p53 knock-out mice (p53-/-) were analyzed by two-dimensional gel electrophoresis (2-DE) and compared with those from p53 wild type (p53+/+) mice. Six types of brain tissue (temporal cortex, cerebellum, hippocampus, striatum, olfactory bulb, and cervical spinal cord) and other control tissues (lung and blood) from 18-week-old non-stress-induced mice were analyzed. The morphology of brains from p53-/- mice appeared to be normal and identical to that of p53+/+ mice, although lungs showed diffuse tumors that may have been caused by p53 deficiency. Comparative 2-D gel analysis showed that, on average, 7 of 886 spots from brain tissue were p53-/- specific, whereas 12 of 1008 spots from lung tissue were p53-/- specific. N-terminal amino acid sequence was determined for p53-/- specific proteins. In all brain tissues from p53-/- mice, a newly identified mouse mitochondrial NADH-ubiquinone oxidoreductase 24 kDa subunit showed decreased expression, and apolipoprotein A1 acidic forms showed increased expression. In addition, brain-type creatine kinase B chain and tubulin beta-5 N-terminal fragment were increased in the p53-/- cerebellum, and a new protein in mouse, hydroxyacylglutathione hydrolase (glyoxalase II) was decreased in the temporal cortex of p53-/- mice. The alterations in protein expression identified in this study may imply a p53-related brain function. This is the first proteomic analysis on the p53-/- mouse brain, and further information based on this study will provide new insights into the p53 function in the CNS.     

Esophageal carcinoma-associated proteins detected by two-dimensional polyacrylamide gel electrophoresis

Patterns of proteins of five surgically resected esophageal carcinomas were studied by two-dimensional polyacrylamide gel electrophoresis with silver staining. The samples of normal esophageal mucosa and esophageal carcinoma from the same patient were compared. Each gel had ca. 300 protein spots and had a similar pattern of proteins. Four spots were observed in all of the esophageal carcinomas that were not present in any of the normal mucosae. The molecular weights and isoelectric points were 46,000 and 5.3, 46,000 and 5.2, 36,000 and 4.7 and 33,000 and 5.1, respectively. One spot was observed in all of the normal mucosae but not in any of the esophageal carcinomas. Its molecular weight and isoelectric point were 27,000 and 5.3, respectively.     

Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis

To increase the number of proteins detectable by two-dimensional electrophoresis (2-DE) in plants, we present a new procedure for extracting total proteins from plant tissue. This method avoids any loss of proteins in the course of sample preparation and results in two different fractions, one comprising mainly the cytoplasmatic proteins, the other one containing predominantly structure bond proteins. 2-DE patterns obtained from these two fractions show that the total number of different protein spots detected exceeds the degree of resolution commonly reported for plant proteins threefold.     

Identification of human myocard proteins separated by two-dimensional electrophoresis.

N-Terminal sequencing, internal sequencing and amino acid analysis were used to identify twelve proteins of the human myocard two-dimensional gel electrophoresis (2-DE) pattern. Amino acid analysis was shown to be a powerful tool in addition to sequencing. The identification of a disease-associated N-terminally blocked protein by internal sequencing was not successful. The twelve identified proteins are the basis of a human myocard 2-DE database.     

Extraction of plant proteins for two-dimensional electrophoresis

Three different extraction procedures for two-dimensional electrophoresis of plant proteins are compared: (i) extraction of soluble proteins with a nondenaturing Tris-buffer, (ii) denaturing extraction in presence of sodium dodecyl sulfate at elevated temperature allowing the solubilization of membrane proteins in addition to a recovery of soluble proteins, and (iii) a trichloroacetic acid-acetone procedure allowing the direct precipitation of total proteins.     

Identification of proteins from human cerebrospinal fluid, separated by two-dimensional polyacrylamide gel electrophoresis

The aim of this work is to display the protein composition of the cerebrospinal fluid by two-dimensional (2-D) gel electrophoresis and identify it using different mass spectrometric techniques. This will enable us to present an overview of the proteins in human cerebrospinal fluid. The comparison of 2-D gels will help us to analyze the normal protein variability in healthy persons and specific protein variations in patients with different neurological diseases (e.g., morbus Alzheimer, chorea Huntington). However, it is not possible to carry out 2-D gel electrophoresis directly with human cerebrospinal fluid due to the high amount of salts, sugars and lipids present. In addition, the total amount of protein is only as high as 0.3-0.7 microg/microL. Therefore, concentration and desalting steps using precipitation and ultrafiltration are necessary. To date we have been able to identify more than 65 spots from 2-D gels using matrix assisted laser desorption/ionization-mass spectrometry and electrospray ionization-mass spectrometry.     

Identification of immunoreactive proteins of Chlamydia trachomatis by Western blot analysis of a two-dimensional electrophoresis map with patient sera.

Western blots of two-dimensional electrophoretic maps of proteins from Chlamydia trachomatis were probed with sera from 17 seropositive patients with genital inflammatory disease. Immunoblot patterns (comprising 28 to 2 spots, average 14.8) were different for each patient; however, antibodies against a spot-cluster due to the chlamydia-specific antigen outer membrane protein-2 (OMP2) were observed in all sera. The next most frequent group of antibodies (15/17; 88%) recognized the hsp60 GroEL-like protein, described as immunopathogenic in chlamydial infections. Reactivity to the major surface-exposed and variable antigen major outer membrane protein (MOMP) was observed at a relatively lower frequency (13/17; 76%). The hsp70 DnaK-like protein was also frequently recognized (11/17; 64.7%) in this patient group. Besides the above confirmatory findings, the study detected several new immunoreactive proteins, with frequencies ranging from 11/17 to 1/17. Some were characterized also by N-terminal amino acid sequencing and homology searches. Amongst these were a novel outer membrane protein (OmpB) and, interestingly, five conserved bacterial proteins: four (23%) sera reacted with the RNA polymerase alpha-subunit, five (29%) recognized the ribosomal protein S1, eight (47%) the protein elongation factor EF-Tu, seven (41%) a putative stress-induced protease of the HtrA family, and seven sera (41%) the ribosomal protein L7/L12. Homologs of the last two proteins were shown to confer protective immunity in other bacterial infections. The data show that immunological sensitization processes commonly thought to play a role in chlamydial pathogenicity may be sustained not only by the hsp60 GroEl-like protein, but also by other conserved bacterial antigens, some of which may be also considered as potential vaccine candidates.     

Proteome analysis of human stomach tissue: separation of soluble proteins by two-dimensional polyacrylamide gel electrophoresis and identification by mass spectrometry

Two-dimensional gel electrophoresis (2-DE) maps for human stomach tissue proteins have been prepared by displaying the protein components of the tissue by 2-DE and identifying them using mass spectrometry. This will enable us to present an overview of the proteins expressed in human stomach tissues and lays the basis for subsequent comparative proteome analysis studies with gastric diseases such as gastric cancer. In this study, 2-DE maps of soluble fraction proteins were prepared on two gel images with partially overlapping pH ranges of 4-7 and 6-9. On the gels covering pH 4-7 and pH 6-9, about 900 and 600 protein spots were detected by silver staining, respectively. For protein identification, proteins spots on micropreparative gels stained with colloidal Coomassie Brilliant Blue G-250 were excised, digested in-gel with trypsin, and analyzed by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-mass spectrometry (DE-MALDI-MS). In all, 243 protein spots (168 spots in acidic map and 75 spots in basic map) corresponding to 136 different proteins were identified. Besides these principal maps, overview maps (displayed on pH 3-10 gels) for total homogenate and soluble fraction, are also presented with some identifications mapped on them. Based on the 2-DE maps presented in this study, a 2-DE database for human stomach tissue proteome has been constructed and is available at http://proteome.gsnu.ac.kr/DB/2DPAGE/Stomach/. The 2-DE maps and the database resulting from this study will serve important resources for subsequent proteomic studies for analyzing the normal protein variability in healthy tissues and specific protein variations in diseased tissues.     

Measurement of specific radioactivity in proteins separated by two-dimensional gel electrophoresis

We report a method to quantify the specific radioactivity of proteins that have been separated by 2-DE. Gels are stained with SyproRuby, and protein spots are excised. The SyproRuby dye is extracted from each spot using DMSO, and the fluorescence is quantified automatically using a plate reader. The extracted gel piece is then dissolved in hydrogen peroxide and radioactivity is quantified by liquid scintillation counting. Gentle agitation with DMSO for 24 h was found to extract all the SyproRuby dye from gel fragments. The fluorescence of the extract was linearly related to the amount of BSA loaded onto a series of 1-D gels. When rat muscle samples were run on 2-DE gels, the fluorescence extracted from 54 protein spots showed a good correlation (r = 0.79, p < 0.001) with the corresponding spot intensity measured by conventional scanning and image analysis. DMSO extraction was found not to affect the amount of radioactive protein left in the gel. When a series of BSA solutions of known specific radioactivity were run on 2-DE gels, the specific radioactivity measured by the new method showed a good correlation (r = 0.98, p < 0.01, n = 5) with the specific radioactivity measured directly before loading. Reproducibility of the method was measured in a series of 2-DE gels containing proteins from the livers of rats and mice that had been injected with [35S]methionine. Variability tended to increase when the amount of radioactivity in the protein spot was low, but for samples containing at least 10 dpm above background the CV was around 30%, which is comparable to that obtained when measuring protein expression by conventional image analysis of SyproRuby-stained 2-DE gels. Similar results were obtained whether spots were excised manually or using a spot excision robot. This method offers a high-throughput, cost-effective and reliable method of quantifying the specific radioactivity of proteins from metabolic labelling experiments carried out in vivo, so long as sufficient quantities of radioactive tracer are used.     

Solubilization of delipidated macrophage membrane proteins for analysis by two-dimensional electrophoresis

Two-dimensional electrophoresis of proteins is often precluded due to the lack of solubilization of cell membrane extracts in an aqueous medium. Various additives and detergents have been used to circumvent the problem, but their efficacy may not be satisfactory. In this study, the removal of lipidic components of the cell membrane extract with chloroform-methanol was used to achieve solubilization. Optimal delipidation was obtained with acetone washings. This procedure increased solubilization of membrane proteins from a murine macrophage cell line, thus showing a substantial improvement in gel resolution. The two-dimensional gels loaded with delipidated extract proved to be free of smearing and horizontal streaking. In addition, other protein spots were revealed that were not detected in the gels loaded with undelipidated cell membrane extract.     

Separation of membrane proteins by two-dimensional electrophoresis using cationic rehydrated strips

Due to their poor solubility during IEF membrane proteins cannot be separated and analyzed satisfactorily with classical 2-DE. A more efficient method for such hydrophobic proteins is the benzyldimethyl-n-hexadecylammonium chloride (16-BAC)/SDS-PAGE, but the corresponding protocol is intricate and time-consuming. We now developed an easy-to-handle electrophoresis method in connection with a novel device which enables reproducible separation of ionic solubilized membrane proteins using individually rehydrated plastic sheet gel strips. These strips are suitable for the first dimension in a 2-D 16-BAC/SDS system and can be handled easily; this is demonstrated by the separation of membrane proteins of human embryonic kidney (HEK293) cells.     

Comparison of ethanol-soluble proteins from different rye (Secale cereale) varieties by two-dimensional electrophoresis

The major storage proteins from six rye varieties, grown under the same conditions in 1997 and 1998 in Rønhave, Denmark, were analyzed by two‐dimensional (2‐D) polyacrylamide gel electrophoresis. The proteins were extracted from ground rye kernels with 70% ethanol and separated by 2‐D electrophoresis. The gels were scanned, compared using ImageMaster® software and the data sets were analyzed by principal component analysis (PCA) using THE UNSCRAMBLER software. Afterwards MATLAB was used to make a cluster analysis of the varieties based on PCA. The analysis of the gels showed, that the protein patterns (number of different proteins and their isoelectric points and molecular weights) from the six rye varieties were different. Based on the presence of unique cultivar‐specific spots it was possible to differentiate between all six varieties if the two harvest years were investigated separately. When the results were combined from the two years five varieties could be differentiated. The results from the PCA confirmed the finding of the unique spots and cluster analysis was made in order to illustrate the results. The combination of the results from 2‐D electrophoresis and other grain characteristics showed that one protein spot was located close to the parameters bread volume and bread height.     

Blotting efficiency investigated by using two-dimensional electrophoresis, hydrophobic membranes and proteins from different sources

Purification and chemical characterization of proteins may be achieved by combining two-dimensional electrophoresis (2-DE) and microsequencing or amino acid analysis. To enable this combination, the protein has to be transferred as completely as possible from the gel into the sequencer. In this study hydrophobic membranes were used as support for the transfer and proteins were transferred from the gels onto the membranes by semidry blotting. Blotting conditions were optimized to obtain high blotting efficiencies for as many proteins of a complex 2-DE pattern as possible. Under optimized conditions, blotting efficiencies between 60% and 100% were obtained for five marker proteins; the mean values from four regions of a 2-DE pattern from 29 unknown proteins of a complex protein mixture from mouse brain were between 60% and 79%. The four commercially available hydrophobic membranes that were compared showed only slight differences in protein amount on the membranes after blotting for whole protein patterns, whereas single proteins occurred with higher amounts on either one or the other membrane. The results of the blotting optimization allowed us to suggest a blotting mechanism with which systematic improvement of the blotting conditions is possible for problematic proteins.     

The establishment of a human liver nuclei two-dimensional electrophoresis reference map

This short communication describes the establishment of a two-dimensional electrophoresis (2-DE) reference map of nuclear proteins isolated from human liver. The human liver nuclei 2-DE reference map contains 1497 spots. In an initial identification study using peptide mass fingerprinting as a means of protein identification we were able to identify 26 spots corresponding to 15 different proteins. The human liver nuclei 2-DE reference map is now included in the SWISS-2DPAGE database, which can be accessed through the ExPASy server (http://www.expasy.ch/ch2d/).     

Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes

While the classical silver stain has been the method of choice for high sensitivity protein visualization on two-dimensional gel electrophoresis (2-D PAGE), post-electrophoretic fluorescent staining with the SYPRO group of dyes has emerged to challenge silver staining for proteome analysis. The latter offers improved sensitivity, higher dynamic range and easy handling. However, most of the published data were derived from analysis of 1-D gel separations. In this work, we have focused on three commercially available fluorescent dyes, SYPRO Ruby, SYPRO Orange and SYPRO Red (Molecular Probes, Eugene, OR, USA) and studied their sensitivity and dynamic range on 2-D PAGE. The use of a multiwavelength fluorescent scanner to image 2-D protein profiles visualized with fluorescent staining is discussed, and a detailed comparison with analysis by silver staining is also provided. These results demonstrate the advantages of using SYPRO dyes, which are in agreement with the literature based on 1-D gel electrophoresis, and give a more realistic understanding of the performance of these fluorescent dyes with 2-D PAGE.     

Characterization of human skin fibroblast extracellular proteins by two-dimensional polyacrylamide gel electrophoresis

Human skin fibroblasts secrete over 50 proteins into the culture medium. In this paper these are characterised using two-dimensional polyacrylamide gel electrophoresis and peptide mapping of proteins metabolically labelled in the presence and absence of tunicamycin. Thirty of these proteins have been shown to be N-glycosides, 4 are O-glycosides and 10 are not glycosylated. Of the major proteins, groups 1-4 have previously been shown to be fibroblast specific. Peptide mapping and tunicamycin treatment has identified that groups 1 and 2, and 3 and 4 are closely related and that groups 1 and 3 arise by N-glycosylation of 2 and 4, respectively. The unglycosylated precursor forms of several other proteins have also been identified. This approach to the analysis of protein secretion provides an abundance of information on many proteins simultaneously and can be used to assess the changes in protein secretion associated with development, and to identify extracellular growth factors and other regulatory proteins.