Two-dimensional analysis of exoproteins of methicillin-resistant Staphylococcus aureus (MRSA) for possible epidemiological applications

We applied two-dimensional gel electrophoresis (2-DE) to the total exoproteins secreted from pathogenic MRSA strains and identified major protein spots by N-terminal amino acid sequence analysis. In approximately 300 to 500 spots visualized on each gel, various exoproteins and cell-associated proteins were identified and their sites on the gels confirmed for construction of a reference map. Major exotoxins such as enterotoxins SEA, SEB, and SEC,, toxic shock syndrome toxin-1 (TSST-1), and hemolysins were distributed in the region of pI 6.8 to 8.1 and MW 21 to 35 kDa. Although the differences between calculated and observed values of pI and MW were relatively small in each exoprotein, those of several proteins including alpha-hemolysin and SEB were considerably deviated from the positions of the expected values. Some exoproteins were detected as multiple spots. These included beta-hemolysin, enterotoxins SEA, SEB, and SEC3, glutamic acid-specific endopeptidase, glycerophosphoryl diester phosphodiesterase and triacylglycerol lipase. The multiple spots of these exoproteins may be generated by the action of own proteases. Certain similarities of 2-DE patterns among strains belonging to the same coagulase types were observed. On the basis of 2-DE image analysis, coagulase type II strains secreted somewhat larger amounts of SEB and SEC3 as well as TSST-1 than the strains belonging to other coagulase types. Taken together, 2-DE analysis of exoproteins is applicable to epidemiological studies for MRSA, as compared with pulsed field gel electrophoresis of restricted chromosomal DNA.     

Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis

2-DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2-DE analysis. Protein samples are spiked with an Alexa-labeled internal standard (ALIS) prior to separation with 2-DE. Due to the high extinction coefficient of the Alexa-fluor, incorporation of 0.1% of total protein is sufficient to allow visualization of the internal standard yet low enough to avoid interference in subsequent quantification and identification steps. Following 2-DE, total proteins are visualized with fluorescent postelectrophoretic stains spectrally separated from ALIS. Four protein stains, Deep Purple, Sulforhodamine G, ruthenium II-tris(bathophenanthroline disulfonate) (RuTBS), and SYPRO Ruby, including improved purification and staining protocols for RuTBS and ten-fold dilutions of SYPRO Ruby were evaluated. All staining protocols were compatible with the ALIS method and had similar LODs (1-4 ng) and dynamic ranges (10(3)). ALIS is a powerful normalization method for quantitative 2-DE which avoids potential problems associated with dual spot migration patterns observed in the DIGE method. Furthermore, ALIS provides significantly improved normality in the distribution of spot abundance-variance compared to normalization through division by the total spot volume.     

Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimensional gel electrophoresis and mass spectrometry

Helicobacter pylori is one of the most common bacterial pathogens and causes a variety of diseases, such as peptic ulcer or gastric cancer. Despite intensive study of this human pathogen in the last decades, knowledge about its membrane proteins and, in particular, those which are putative components of the type IV secretion system encoded by the cag pathogenicity island (PAI) remains limited. Our aim is to establish a dynamic two-dimensional electrophoresis-polyacrylamide gel electrophoresis (2-DE-PAGE) database with multiple subproteomes of H. pylori (http://www.mpiib-berlin.mpg.de/2D-PAGE) which facilitates identification of bacterial proteins important in pathogen-host interactions. Using a proteomic approach, we investigated the protein composition of two H. pylori fractions: soluble proteins and structure-bound proteins (including membrane proteins). Both fractions differed markedly in the overall protein composition as determined by 2-DE. The 50 most abundant protein spots in each fraction were identified by peptide mass fingerprinting. We detected four cag PAI proteins, numerous outer membrane proteins (OMPs), the vacuolating cytotoxin VacA, other potential virulence factors, and few ribosomal proteins in the structure-bound fraction. In contrast, catalase (KatA), gamma-glutamyltranspeptidase (Ggt), and the neutrophil-activating protein NapA were found almost exclusively in the soluble protein fraction. The results presented here are an important complement to genome sequence data, and the established 2-D PAGE maps provide a basis for comparative studies of the H. pylori proteome. Such subproteomes in the public domain will be effective instruments for identifying new virulence factors and antigens of potential diagnostic and/or curative value against infections with this important pathogen.     

A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard

The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression. Conventional methods rely on comparing images from at least 2 different gels. Due to the high variation between gels, detection and quantification of protein differences can be problematic. Two-dimensional difference gel electrophoresis (Ettan trade mark DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel. Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel. The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.     

Improved comparative proteome analysis based on two-dimensional gel electrophoresis

The purpose of this study was to test the extent to which differences in spot intensity can be reliably recognized between two groups of two-dimensional electrophoresis gels (pH 4-7, visualized with ruthenium fluorescent stain) each loaded with different amounts of protein from rat brain (power analysis). Initial experiments yielded only unsatisfactory results: 546 spots were matched from two groups of 6 gels each loaded with 200 microg and 250 microg protein, respectively. Only 72 spots were higher (p<0.05), while 58 spots were significantly lower in the 250-microg group. The construction of new apparatuses that allowed the simultaneous processing of 24 gels throughout all steps between rehydration and staining procedure considerably lowered the between-gel variation. This resulted in the detection of significant differences in spot intensities in 77-90% of all matched spots on gel groups with a 25% difference in protein load. This applied both when protein from 24 biological replicates was loaded onto two groups of 12 gels and when two pooled tissue samples were each loaded onto 6 gels. At a difference of 50% in protein load, more than 90% of all spots differed significantly between two experimental groups.     

Plasma proteomics of lung cancer by a linkage of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis

To investigate aberrant plasma proteins in lung cancer, we compared the proteomic profiles of serum from five lung cancer patients and from four healthy volunteers. Immuno-affinity chromatography was used to deplete highly abundant plasma proteins, and the resulting plasma samples were separated into eight fractions by anion-exchange chromatography. Quantitative protein profiles of the fractionated samples were generated by two-dimensional difference gel electrophoresis, in which the experimental samples and the internal control samples were labeled with different dyes and co-separated by two-dimensional polyacrylamide gel electrophoresis. This approach succeeded in resolving 3890 protein spots. For 364 of the protein spots, the expression level in lung cancer was more than twofold different from that in the healthy volunteers. These differences were statistically significant (Student's t-test, p-value less than 0.05). Mass spectrometric protein identification revealed that the 364 protein spots corresponded to 58 gene products, including the classical plasma proteins and the tissue-leakage proteins catalase, clusterin, ficolin, gelsolin, lumican, tetranectin, triosephosphate isomerase and vitronectin. The combination of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis provides a valuable tool for serum proteomics in lung cancer.     

A novel approach to spot detection for two-dimensional gel electrophoresis images using pixel value collection

Separation of complex mixtures of proteins by two-dimensional gel electrophoresis (2-DE) is a fundamental component of current proteomic technology. Quantitative analysis of the images generated by digitization of such gels is critical for the identification of alterations in protein expression within a given biological system. Despite the availability of several commercially available software packages designed for this purpose, image analysis is extremely resource intensive, subjective and remains a major bottleneck. In addition to reducing throughput, the requirement for manual intervention results in the introduction of operator subjectivity, which can limit the statistical significance of the numerical data generated. A key requirement of image analysis is the accurate definition of protein spot boundaries using a suitable method of image segmentation. We describe a method of spot detection applicable to 2-DE image files using a segmentation method involving pixel value collection via serial analysis of the image through its range of density levels. This algorithm is reproducible, sensitive, accurate and primarily designed to be automatic, removing operator subjectivity. Furthermore, it is believed that this method may offer the potential for improved spot detection over currently available software.     

Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry

Exosomes are 40-100 nm vesicles released by numerous cell types and are thought to have a variety of roles depending on their origin. Exosomes derived from antigen presenting cells have been shown to be capable of initiating immune responses in vivo and eradicating established tumours in murine models. Tumour-derived exosomes can be utilised as a source of tumour antigen for cross-priming to T-cells and are thus of interest for use in anti-tumour immunotherapy. Further exploration into the protein composition of exosomes may increase our understanding of their potential roles in vivo and this study has examined the proteome of exosomes purified from cell supernatants of the melanoma cell lines MeWo and SK-MEL-28. The vesicular nature and size (30-100 nm) of the purified exosomes was confirmed by electron microscopy and sucrose density gradient centrifugation. Western blotting demonstrated the absence of calnexin and cytochrome c, verifying the purity of the exosome preparations, as well as enrichment of MHC class I and the tumour-associated antigens Mart-1 and Mel-CAM. The two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein profiles of exosomes from the two cell lines were highly comparable and strikingly different from the profiles of the total cell lysates. Mass spectrometric sequencing identified proteins present in 49 protein spots in the exosome lysates. Several of these have been identified previously in exosomes but some are novel, including p120 catenin, radixin, and immunoglobulin superfamily member 8 (PGRL). Proteins present in whole-cell lysates that were significantly reduced or excluded from exosomes were also identified and included several mitochondrial and lysosomal proteins, again confirming the proposed endosomal origin of exosomes. This study presents a starting point for future more in-depth protein studies of tumour-derived exosomes which will aid the understanding of their biogenesis and targeting for use in anti-tumour immunotherapy protocols.     

Improvement of two-dimensional gel electrophoresis proteome maps of the haloarchaeon Haloferax volcanii

Proteins of haloarchaea are remarkably unstable in low-ionic-strength solvents and tend to aggregate under standard two-dimensional (2-D) gel electrophoresis conditions, causing strong horizontal streaking. We have developed a new approach to generate 2-D maps of halophilic proteins which included washing cells with 1.5 M Tris-HCl buffer. In addition, proteins were precipitated with acetone, solubilized with urea and thiourea in the presence of the sulfobetaine detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS), reduced with tributylphosphine (TBP), and separated with microrange strips of immobilized pH gradients (pH 3.9-5.1). This combination enabled the construction of highly reproducible 2-D maps of Haloferax volcanii proteins.     

Proteomic analysis of human very low-density lipoprotein by two-dimensional gel electrophoresis and MALDI-TOF/TOF

Biochemical studies of lipoproteins have shed light on their composition, highly contributing to the comprehension of their function. Due to the complexity of their structure, however, an in-depth structural analysis, in terms of components and PTMs, may still unravel important players in physiological and pathological processes of lipid metabolism. In this study, we performed a protein map of very low-density lipoprotein (VLDL) using a 2-DE MALDI-TOF/TOF proteomic approach. Several VLDL-associated apolipoproteins were identified, including five isoforms of apoE, three isoforms of apoC-IV, and one isoform each of apoC-III, apoM, apoA-I, and apoA-IV. Notably, we also identified seven isoforms of apoL-I and two isoforms of prenylcysteine lyase as new VLDL-associated proteins. Furthermore, we were able to identify PTM of apoE, which was found to be differently O-glycosylated at Thr212 residue, and PTM of apoL-I which we described, for the first time, to be phosphorylated at Ser296. While the physiological relevance of our finding remains to be assessed, we believe that our results will be useful as reference for future studies of VLDL structure in specific physiopathological conditions.     

镉胁迫下三个萝卜栽培种蛋白质变化的双向电泳比较研究

在0.5mmol·L^-1 Cd²⁺处理时,小五缨(XWY)的发芽率下降至92%,并且随着Cd²⁺浓度的增加,发芽率逐渐降低,在1和5mmol·L^-1 Cd²⁺浓度处理时,发芽率分别降至83%和67%。象牙白(XYB)则在5mmol·L^-1 Cd²⁺浓度时,发芽率下降。卫青(WQ)在0.05mmol·L^-1 Cd²⁺浓度时,发芽率已降至83%, 1和5mmol·L^-1时则下降至58%。幼苗生长也明显受Cd²⁺处理影响,在0.05mmol·L^-1 Cd²⁺处理时, 3种栽培萝卜的幼苗生长均受到明显影响,并且随Cd²⁺浓度的增加,生长受抑加重。从发芽率和幼苗生长两种实验结果看,卫青对Cd²⁺最为敏感。双向电泳结果表明, Cd²⁺处理后3种栽培萝卜幼苗中蛋白质组分有明显变化。小五缨中, 0.1 mmol·L^-1Cd²⁺处理后,有5个蛋白质点消失, 15个新的蛋白质点被诱导产生。象牙白中, 2个蛋白质点消失, 1个蛋白质点含量减少, 13个新的蛋白质点被诱导产生。卫青中, 12个新的蛋白质点诱导产生,但没有发现蛋白质点消失现象。Cd²⁺处理后, 3种栽培萝卜中,蛋白质合成的变化与幼苗生长受抑存在明显相关性,这一实验结果对于探讨萝卜Cd²⁺害的生化机理是有重要意义的。     

Reduction of proteins during sample preparation and two-dimensional gel electrophoresis of woody plant samples

Protein extraction procedure and the reducing agent content (DTT, dithioerythritol, tributyl phosphine and tris (2-carboxyethyl) phosphine (TCEP)) of the sample and rehydration buffers were optimised for European beech leaves and roots and Norway spruce needles. Optimal extraction was achieved with 100 mM DTT for leaves and needles and a mixture of 2 mM TCEP and 50 mM DTT for roots. Performing IEF in buffers containing hydroxyethyldisulphide significantly enhanced the quality of separation for all proteins except for acidic root proteins, which were optimally focused in the same buffer as extracted.     

A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure

We report the results of a systematic investigation to quantify the losses of protein during a well-established two-dimensional polyacrylamide gel electrophoresis (2-DE) procedure. Radioactively labelled proteins ([(14)C]bovine serum albumin and a homogenate prepared from the liver of a rat that had been injected with [(35)S]methionine) were used, and recovery was quantified by digesting pieces of gel in H(2)O(2) and subjecting the digests to liquid scintillation counting. When samples were loaded onto the first dimension immobilised pH gradient strips by in-gel rehydration, recovery of protein from the strips was 44-80% of the amount of protein loaded, depending on the amount of protein in the sample. Most of the unrecovered protein appeared to have adhered to the reswelling tray. Losses during isoelectric focusing (IEF) were much smaller (7-14%), although approximately 2% of the protein appeared to migrate from sample strips to adjacent blank strips in the focussing apparatus. A further 17-24% of the proteins were lost into the buffers during equilibration prior to running in the second dimension. Losses during the second dimension run and subsequent staining with SYPRO Ruby amounted to less than 10%. The overall loss during 2-DE was reduced by approximately 25% when proteins were loaded onto the IEF strips using sample cups instead of by in-gel rehydration. These extensive and variable losses during the 2-DE procedure mean that spot intensities on 2-DE gels cannot be used to derive reliable, quantitative information on the amounts of proteins present in the original sample.     

The whereabouts of transmembrane proteins from rat brain synaptosomes during two-dimensional gel electrophoresis

Little is known about what happens to transmembrane proteins (TMP) in 2-DE. In order to obtain more insight into the whereabouts of these proteins we prepared membrane-enriched synaptosomes from rat frontal cortex and washed them with 7 M urea or Na(2)CO(3). From each preparation, 200 microg protein was loaded on 2-DE gels covering the 4-7 and 6-11 pH ranges, respectively. MALDI-MS/MS analysis detected only 3 TMP among 421 identified spots. However, when the samples had been washed with Na(2)CO(3), only few well-focused spots remained detectable on the gel covering the pH 6-11 range. Instead, a heavily ruthenium-stained smear became visible at the upper edge of the gel at the location where the samples had been applied by cup loading. LC-MS/MS analysis revealed that this smear contained 38 unfocused TMP with up to 12 transmembrane helices. After transfer to the second dimension, no major areas of protein staining were left on the IPG strips. This indicates that after extraction and denaturation the TMP may form high-molecular aggregates, due to their "hydrophobic interactions". These aggregates enter the IPG strips, but do not focus regularly. They are then transferred onto the 2-DE-gels, where they remain caught at the upper edge.     

Increasing the resolution of polyacrylamide gel electrophoresis by varying the degree of gel crosslinking

Resolution of polyacrylamide gel electrophoresis may be substantially improved by taking advantage of the gel sieving effects of varying concentrations of bisacrylamide crosslinker. A dilution procedure is described which permits simultaneous variation of both total acrylamide concentration and percent crosslinking within a single linear regression analysis.This work was supported by NSF Grant 10584 and NIH Grant 23504.     

Proteome analysis of glandular parotid and submandibular-sublingual saliva in comparison to whole human saliva by two-dimensional gel electrophoresis

The secretions of the salivary parotid and submandibular-sublingual (SMSL) glands constitute the main part of whole human saliva (WS) in which proline-rich proteins (PRPs) and mucins represent dominant groups. Although proteome analysis had been performed on WS, no identification of PRPs or mucins by 2-DE and MS was achieved in WS and no comprehensive analysis of both glandular secretions is available so far. The aim of this study was to compare the protein map of WS to parotid and SMSL secretions for the display of PRPs and mucins. WS and glandular secretions were subjected to 2-DE and spots were analyzed by MALDI-MS. New components identified in WS were cyclophilin-B and prolyl-4-hydroxylase. Also acidic and basic PRPs as well as the proline-rich glycoprotein (PRG) could now be mapped in WS. Acidic PRPs were found equally in parotid and SMSL secretions, whereas basic PRPs and PRG were found primarily in parotid secretion. Salivary mucin MUC7 was identified in SMSL secretion. Thus, the more abundant proteins of WS can be explained mainly by mixed contributions of parotid and SMSL secretions with only few components remaining that may be derived from local sources in the oral cavity.     

Two-dimensional polyacrylamide gel electrophoresis of membrane proteins

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the protein, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity, antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer at a low level.     

Direct chemiluminescent imaging detection of human serum proteins in two-dimensional polyacrylamide gel electrophoresis

A novel chemiluminescence (CL)-based imaging method capable of directly detecting proteins in polyacrylamide gels after electrophoresis is proposed. Human serum proteins are presently detected by a direct CL imaging method after native 2-D PAGE. As a consequence, some proteins, including haptoglobin (Hp), Hp precursor, hemopexin (Hpx) precursor, Ig alpha-1 chain C region, and Complement C3 precursor can be detected and identified by MS and MS/MS techniques. These proteins are all acute phase proteins, which have been defined as biomarkers for certain diseases. Moreover, serum proteins from healthy people and cirrhotic patients were analyzed. A decrease in Hp spots for cirrhotic patients could be confirmed. The CL imaging conditions were optimized, including the concentrations of H(2)O(2) and luminol. The process of CL detection of proteins is simple, and there is no need for specialized equipment. In comparison with the traditional CBB-R250 staining method, the detection sensitivity was improved and the detection period decreased about 70 times. Hence, this technique possesses potentials as a rapid, convenient, and inexpensive analytical technique for protein detection and for the diagnosis of diseases.     

Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dye

To identify the proteomic alterations associated with carcinogenesis of hepatocellular carcinoma (HCC), we compared the protein expression profiles of nine HCC cell lines with those of primary cultured hepatocytes established from five individuals. A differential proteomic study was performed by two-dimensional difference gel electrophoresis, in which protein samples are labeled with different fluorescent dyes and separated according to their isoelectric point and molecular weight. To label the protein samples, we used a newly developed and highly sensitive fluorescent dye, which reacts with all reduced cysteine residues of proteins. Principal component analysis based on the intensity of 1238 protein spots indicated that the HCC cells and the normal hepatocytes had distinct proteomic profiles. The Wilcoxon test was used to determine the protein spots whose intensity was differentially regulated in the HCC cells compared with the normal hepatocytes, and mass spectrometric analysis was used to identify the proteins corresponding to the spots. The proteins identified are involved in cell cycle regulation, binding to a tumor-suppressor gene product, fatty acid binding, and regulation of translation. Western blotting with specific antibodies revealed the overexpression of PCNA, EB1 and E-FABP in HCC tissues compared with noncancerous tissues. Aberrant regulation of EB1 and E-FABP has not previously been implicated in the development of HCC.     

A multifaceted analysis of viperid snake venoms by two-dimensional gel electrophoresis: an approach to understanding venom proteomics

The complexity of Viperid venoms has long been appreciated by investigators in the fields of toxinology and medicine. However, it is only recently that the depth of that complexity has become somewhat quantitatively and qualitatively appreciated. With the resurgence of two-dimensional gel electrophoresis (2-DE) and the advances in mass spectrometry virtually all venom components can be visualized and identified given sufficient effort and resources. Here we present the use of 2-DE for examining venom complexity as well as demonstrating interesting approaches to selectively delineate subpopulations of venom proteins based on particular characteristics of the proteins such as antibody cross-reactivity or enzymatic activities. 2-DE comparisons between venoms from different species of the same genus (Bothrops) of snake clearly demonstrated both the similarity as well as the apparent diversity among these venoms. Using liquid chromatography/tandem mass spectrometry we were able to identify regions of the two-dimensional gels from each venom in which certain classes of proteins were found. 2-DE was also used to compare venoms from Crotalus atrox and Bothrops jararaca. For these venoms a variety of staining/detection protocols was utilized to compare and contrast the venoms. Specifically, we used various stains to visualize subpopulations of the venom proteomes of these snakes, including Coomassie, Silver, Sypro Ruby and Pro-Q-Emerald. Using specific antibodies in Western blot analyses of 2-DE of the venoms we have examined subpopulations of proteins in these venoms including the serine proteinase proteome, the metalloproteinase proteome, and the phospholipases A2 proteome. A functional assessment of the gelatinolytic activity of these venoms was also performed by zymography. These approaches have given rise to a more thorough understanding of venom complexity and the toxins comprising these venoms and provide insights to investigators who wish to focus on these venom subpopulations of proteins in future studies.