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.     

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.     

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.     

A reliable two-dimensional gel electrophoresis procedure for separating neural proteins

A facile two-dimensional gel electrophoresis procedure has been developed for the analysis of neural tissue proteins which eliminates the serious problems associated with protein insolubility at the point of sample application onto polymerized first-dimension isoelectric focusing gels. This was accomplished by combining the methods of two previously published procedures. Our procedure provides an alternative method to the complex gel systems often employed for less soluble proteins, and yields very reproducible, high resolution separations. This procedure, which is in routine use in our laboratories for the analysis of total proteins extracted from retina and brain, produces protein patterns that are easily compared using both visual and computer-assisted image analysis techniques. Presented here are the results of a set of experiments designed to identify proteins unique to retina. This procedure should be useful to investigators studying protein changes resulting from genetic mutation, development, drug treatment or disease, in neural tissue as well as in virtually all other tissues.     

New physically adsorbed polymer coating for reproducible separations of basic and acidic proteins by capillary electrophoresis

In this work, a new physically adsorbed coating for capillary electrophoresis (CE) is presented. The coating is based on a N,N-dimethylacrylamide-ethylpyrrolidine methacrylate (DMA-EPyM) copolymer synthesized in our laboratory. The capillary coating is simple and easy to obtain as only requires flushing the capillary with a polymer aqueous solution for 2 min. It is shown that by using these coated capillaries the electrostatic adsorption of a group of basic proteins onto the capillary wall is significantly reduced allowing their analysis by CE. Moreover, the DMA-EPyM coating provides reproducible separations of the basic proteins with RSD values for migration times lower than 0.75% for the same day (n = 5) and lower than 3.90% for three different days (n = 15). Interestingly, the electrical charge of the coated capillary wall can be modulated by varying the pH of the running buffer which makes possible the analysis of basic and acidic proteins in the same capillary. The usefulness of this coating is further demonstrated via the reproducible separation of whey (i.e. acidic) proteins from raw milk. The coating protocol should be compatible with both CE in microchips and CE-MS of different types of proteins.     

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.     

Automated chip-based nanoelectrospray-mass spectrometry for rapid identification of proteins separated by two-dimensional gel electrophoresis

We report a method using a fully automated chip-based nanoelectrospray system for two-dimensional (2-D) gel sample analyses with mass spectrometric detection. The automated nanoelectrospray system, consisting of the NanoMate and electrospray ionization (ESI) chip, serves as both an autosampler and nanoESI source. This infusion system aspirates samples from a 96-well plate using disposable pipette tips and then delivers these samples sequentially to an ESI chip. This chip is a fully integrated monolithic device consisting of a 10x10 array of nozzles. The automated nanoelectrospray system is easily controlled through software, permitting the user to select the number of samples to be analyzed, the volume of sample to aspirate, the spray voltage, and analysis time. The system offers all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without analyte carryover. The system was used for a protein identification study of 2-D gel spots of both Escherichia coli and yeast crude cell extracts. The identification of 50 spots from E. coli crude cell extract and 27 spots from yeast extract is presented, demonstrating the powerful combination of the automated nanoESI system, the Thermo Finnigan LCQ Deca ion-trap mass spectrometer, and SEQUEST search software. In addition, the effects of silver staining and colloidal Coomassie blue staining of 2-D gel spots on the detection sensitivity and protein sequence coverage are compared and discussed. Furthermore, the comparison results using the multiwell microscale preparation kit versus manual extraction for in-gel samples are presented.     

Quantitative two-dimensional gel electrophoresis analysis of human fibroblasts transformed by ras oncogenes.

Quantitative two-dimensional gel electrophoresis was used to compare the cellular protein patterns of a normal foreskin-derived human fibroblasts cell line (LG1) and three immortal derivatives of LG1. One derivative, designated MSU-1.1 VO, was selected for its ability to grow in the absence of serum and is non-tumorigenic in athymic mice. The other two strains were selected for focus-formation following transfection with either Ha-ras or N-ras oncogenes and form high grade malignant tumors. Correspondence and cluster analysis provided a nonbiased estimate of the relative similarity of the different two-dimensional patterns. These techniques separated the gel patterns into three distinct classes: LG1, MSU-1.1 VO, and the ras transformed cell strains. The MSU-1.1 VO cells were more closely related to the parental LG1 than to the ras-transformed cells. The differences between the three classes were primarily quantitative in nature: 16% of the spots demonstrated statistically significant changes (P < 0.01, T test, mean ratio of intensity > 2) in the rate of incorporation of radioactive amino acids. The patterns from the two ras-transformed cell strains were similar, and variations in the expression of proteins that occurred between the separate experiments obscured consistent differences between the Ha-ras and N-ras transformed cells. However, while only 9 out of 758 spots were classified as different (1%), correspondence analysis could consistently separate the two ras transformants. One of these spots was five times more intense in the Ha-ras transformed cells than the N-ras.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Study of grasshopper meiosis-associated proteins using two-dimensional polyacrylamide gel electrophoresis

Premeiotic and meiotic whole testes from grasshoppers were compared for the presence of meiosis associated proteins using one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. One-dimensional sodium dodecyl sulphate-polyacrylamide gels detected differences between premeiotic and meiotic samples but two-dimensional gels gave more precise results. Isoelectric focusing revealed only one meiosis-associated protein, while nonequilibrium pH gradient electrophoresis detected five more. It is not known whether these proteins relate to the nuclear aspects of meiosis, or associated cellular changes. These proteins have been electrophoretically purified and monoclonal antibodies are being prepared.     

A general method for the rapid characterization of tyrosine-phosphorylated proteins by mini two-dimensional gel electrophoresis

Our preliminary results are reported in the investigation of the tyrosine phosphorylation cascade triggered by the stimulation of the insulin receptor in the adipocyte cell line 3T3-L1 using a mini two-dimensional gel electrophoresis approach. The minigel format, 8 x 10 cm, was found sufficiently resolving and reproducible to study complex biological samples while considerably increasing throughput and lowering costs compared to larger gel formats. Consequently, we used the minigel format to rapidly screen a large number of samples, of which only the most relevant were then analyzed by optimized, preparative two-dimensional gels. The accurate localization and relative quantification of tyrosine-phosphorylated proteins was performed using a nonradioactive triple labeling method. After transfer onto polyvinylidene difluoride (PVDF) membranes, proteins were stained with Sypro Ruby to verify the separation quality and to localize the general region of interest for immunostaining. The membranes were subsequently blocked with polyvinylpyrrolidone-40 and probed with the relevant antibodies for visualization of the phosphorylated proteins by chemiluminescence. Finally, membranes were stained with colloidal gold to obtain a pattern reminiscent of the silver staining of a polyacrylamide gel. We believe that the presented strategy can be generalized for any gel application in which a protein has to be detected and identified based on its immunoreactivity.     

Toward a steady-state pore limit electrophoresis dimension for native proteins in two-dimensional polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis in linear pore gradients (4.8 to 48% T, 5% CBis) provides for migration arrest, in a practical sense, after about 5000 Vh for proteins of 290 and 450 kDa, but not for smaller proteins over 20,000 Vh. The arrest is not due to inadequate field strength nor is it caused by water redistribution within pore gradient gels. The possibility is being discussed that exponential pore gradients, and a higher or a lower degree of crosslinking suggested by the literature may be remedies for the present failure to arrest the migration of smaller proteins.     

A novel probe for chemiluminescent image detection of proteins in two-dimensional gel electrophoresis

Carrier ampholytes were found to enhance the chemiluminescence (CL) emission from the 3-aminophthalic hydrazide (luminol)-hydrogen peroxide system. They can be used as a chemiluminescent probe for rapid detection of major proteins in gels. This probe attracted much interest due to its ability to attach proteins, and to the possibility to combine it with separation techniques generating the CL emission directly. Increased signal intensity was achieved employing optimized concentrations of the carrier ampholyte enhancer. The binding of carrier ampholyte to proteins was found to occur at the pI of the proteins. Proteins from different regions of the gels were identified by their matrix-assisted TOF mass spectra and by appropriate database search, the results illustrating the possibility of major protein detection in human serum. Direct CL image detection with the carrier ampholyte probe can be applied for the detection of characteristic proteins in patients, i.e., proteins which cannot be detected without the probe.     

Degradation of individual intracellular proteins analyzed by two-dimensional gel electrophoresis and computerized video densitometry

A technique is described for the analysis of degradation rates of individual intracellular proteins, based on pulse-chase-labeling of cells using radioactive amino acids [35S]methionine, two-dimensional polyacrylamide gel electrophoresis, fluorography and scanning of the fluorograms by a computerized video densitomter. As compared to scintillation counting of individual protein spots resolved by two-dimensional gel electrophoresis, this method allows a rapid and precise determination of the degradation rates of individual intracellular proteins. In the present study, degradation rates of individual intracellular proteins of normal human skin fibroblasts and skin fibroblasts from patients with Duchenne muscular dystrophy were compared. Rates of degradation for proteins PIIa, PIIb and PIIc recently described as cell-type-specific proteins were significantly enhanced (p less than 0.01) in fibroblast cultures of Duchenne muscular dystrophy origin.     

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.     

A simple, inexpensive method for the salt-free concentration of proteins for analysis by two-dimensional gel electrophoresis

A rapid, inexpensive method for the salt-free concentration of small quantities of proteins for analysis by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been developed. Proteins adsorbed to diatomaceous earth are subsequently removed using either sodium dodecyl sulfate or urea solubilization reagents for 2D-PAGE analysis. This procedure has been found to concentrate proteins having wide ranges of molecular weight and charge. It is also valuable for the concentration of large numbers of small samples from cells cultured in vitro.     

Quantitative detection of phosphoproteins by combination of two-dimensional difference gel electrophoresis and phosphospecific fluorescent staining

Here we combine a standard two-dimensional difference gel electrophoresis (DIGE) protocol with subsequent post-staining of gels with phosphospecific fluorescent Pro-Q Diamond dye. The combination of these two methods for fluorescence detection of proteins allows quantitative detection of phosphoproteins in 2-DE-gels. We established this protocol within a functional proteomics experiment. Mammary epithelial cells (EpH4) were stimulated in culture by epidermal growth factor (EGF), endosomal fractions prepared after subcellular fractionation and phosphorylated proteins successfully detected on endosomes. For instance, Endo A cytokeratin, known as phosphoprotein and differentiation marker inducible by MAPK signaling, was identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). With this protocol, all steps of combined proteome and phosphoproteome profiling experiments are significantly simplified and accelerated, taking full advantage of both methods in terms of specificity, sensitivity and accuracy of quantification.     

An immobiline DryStrip application method enabling high-capacity two-dimensional gel electrophoresis

In the field of proteomics the need to detect low-abundance cellular components, such as regulatory proteins, is of critical importance. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is one of the most commonly used separation tools for these biological investigations. In this paper we report an alternative micropreparative 2-D PAGE sample application method, called the "paper bridge loading" method. This method makes it possible to apply a larger sample volume to commercially available immobilized pH gradient (IPG) strips. The Vh products required for focusing are only marginally longer than those used in analytical experiments. The method was compared to traditional cup loading and in-gel rehydration. With 18 cm long narrow-range Immobiline DryStrip pH 4.5-5.5, the "paper bridge" method allowed the application of 10 mg human plasma proteins compared to 3 mg with traditional loading methods. The corresponding figures using Escherichia coli sample was found to be 6 mg and less than 2 mg, respectively. The paper bridge method also showed the best results in terms of spot resolution and separation of high molecular weight proteins.     

Spot overlapping in two-dimensional polyacrylamide gel electrophoresis separations: a statistical study of complex protein maps

A statistical approach able to extract the information contained in a two-dimenisional polyacrylamide gel electrophoresis (2-D PAGE) separation is here reported. The method is based on the quantitative theory of peak overlapping, a procedure previously developed by the authors and here extended to 2-D separations. The whole map is divided into many strips in order to obtain 1-D separations on which the statistic procedure is applied: the developed algorithms, on the basis of spot experimental data (intensity and spatial coordinates) permit to estimate the intrinsic number of components and to single out the specific order present in spot positions. The procedure was validated on computer-simulated maps. Its applicability to real samples was tested on maps obtained from literature sources. The following important information on protein mixtures can be extracted: (i) the number of proteins can be accurately estimated, on the basis of the spatial coordinates and intensities of spots detected in the 2-D PAGE map; (ii) the model describing distribution of interdistance between adjacent spots can be identified in both the separation dimensions; (iii) the presence of repeated interdistances in spot positions in the maps can be easily singled out: these regularities suggest specific protein modifications.