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1.
2-D separation of bacterial membrane proteins is still difficult despite using high-resolution IPG-IEF/SDS-PAGE. We were searching for alternative methods to avoid typical problems such as precipitation, low solubility, and aggregation of membrane proteins in the 1-D separation with IPG-IEF. Blue native electrophoresis (BNE) and agarose IEF (A-IEF) were tested for their separation capacity and their capability of replacing IPG-IEF in the first dimension. SDS-PAGE was chosen for the second dimension on account of its outstanding resolution. We could confirm that only A-IEF was a useful replacement for the IPG-IEF in the first dimension resulting in 2-D protein distributions with additional membrane protein spots not being found after IPG-IEF/SDS-PAGE. A second interesting result was that the agarose IEF mediates the possibility of separation of membrane proteins in a partially native state in the first dimension. This native A-IEF resulted in drastically changed spot patterns with an acidic shift of nearly all spots and divergent distribution of proteins compared to non-native A-IEF and IPG-IEF. We found out that native and non-native A-IEF are powerful tools to supplement IPG-IEF/SDS-PAGE. 相似文献
2.
Two-dimensional gel electrophoresis maps of the proteome and phosphoproteome of primitively cultured rat mesangial cells 总被引:1,自引:0,他引:1
Mesangial cells (MC) play an important role in maintaining the structure and function of the glomerulus. The proliferation of MC is a prominent feature of many kinds of glomerular disease. The first reference 2-DE maps of rat mesangial cells (RMC), stained with silver staining or Pro-Q Diamond dye, have been established here to describe the proteome and phosphoproteome of RMC, respectively. A total of 157 selected protein spots, corresponding to 118 unique proteins, have been identified by MALDI-TOF-MS or LC-ESI-IT-MS/MS, in which 37 protein spots representing 28 unique proteins have also been stained with Pro-Q Diamond, indicating that they are in phosphorylated forms. All the identified proteins were bioinformatically annotated in detail according to their physiochemical characteristics, subcellular location, and function. Most of the separated or identified protein spots are distributed in the area of mass 10-70 kDa and pI 5.0-8.0. The identified proteins include mainly cytoplasmic and nuclear proteins and some mitochondrial, endoplasmic reticulum, and membrane proteins. These proteins are classified into different functional groups such as structure and mobility proteins (21.2%), metabolic enzymes (16.9%), protein folding and metabolism proteins (13.6%), signaling proteins (14.4%), heat-shock proteins (7.6%), and other functional proteins (12.7%). While structure and mobility proteins are mostly represented by protein spots with high abundance, signaling proteins are mostly represented by protein spots with relatively low abundance. Such a 2-DE database for RMC, especially with many signaling proteins and phosphoproteins characterized, will provide a valuable resource for comparative proteomics analysis of normal and pathologic conditions affecting MC function or pathologic progress. 相似文献
3.
Two-dimensional (2-D) Blue Native/SDS gel electrophoresis combines a first-dimensional separation of monomeric and multimeric proteins in their native state with a second denaturing dimension. These high-resolution 2-D gels aim at identifying multiprotein complexes with respect to their subunit composition. We applied this method for the first time to analyze two human platelet subproteomes: the cytosolic and the microsomal membrane protein fraction. Solubilization of platelet membrane proteins was achieved with the nondenaturing detergent n-dodecyl-beta-D-maltoside. To validate native solubilization conditions, we demonstrated the correct assembly of the Na,K-ATPase, a functional multimeric transmembrane protein, when expressed in Xenopus oocytes. We identified 63 platelet proteins after in-gel tryptic digestion of 58 selected protein spots and liquid chromatography-coupled tandem mass spectrometry. Nine proteins were detected for the first time in platelets by a proteomic approach. We also show that this technology efficiently resolves several known membrane and cytosolic multiprotein complexes. Blue Native/SDS gel electrophoresis is thus a valuable procedure to analyze specific platelet subproteomes, like the membrane(-bound) protein fraction, by mass spectrometry and immunoblotting and could be relevant for the study of protein-protein interactions generated following platelet activation. 相似文献
4.
Study of the development of thermoresistance in human pancreatic carcinoma cell lines using proteome analysis 总被引:3,自引:0,他引:3
In order to find candidate proteins that are potentially associated with the thermoresistant phenotype in combination with drug resistance, we analyzed the differential protein expression in vitro in the human pancreatic cancer cell line EPP85-181-P and classical and atypical multidrug-resistant variants and their thermoresistant counterparts using proteomics. This study identifies sets of proteins that may lead to the development of thermoresistance. These results provide a fundamental basis to elucidate the molecular mechanism of thermoresistance and chemoresistance phenomena that may assist the therapy of inoperable cancers. 相似文献
5.
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. 相似文献
6.
7.
应用双向凝胶电泳(2-DE)结合质谱技术研究饥饿诱导自噬的HeLa细胞(实验组)与正常培养的HeLa细胞(对照组)的差异表达蛋白质。结果显示对照组样本的2-DE图谱检测到1253±100个蛋白斑点,实验组样本检测到1216±125个蛋白斑点,二者主要斑点的位置与数量基本一致。对差异表达蛋白进行质谱分析,首次发现前折叠素2,转胶蛋白2,乳腺癌扩增序列2和Annexin A3在自噬细胞中表达上调,提示这4种蛋白可能参与饥饿诱导的细胞自噬。本研究为自噬的机制初步提供了线索。 相似文献
8.
Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis 总被引:1,自引:0,他引:1
We previously demonstrated the separation of proteins by isoelectric focusing (IEF) over pH 4-8 immobilized pH gradients (IPGs) over 54 cm (Poland et al., Electrophoresis 2003, 24, 1271). Here we show that similar results can be conveniently achieved using commercially available IPGs of appropriate pH ranges positioned end-on-end in series during electrophoresis, which we term "daisy chain IEF". Proteins efficiently electrophorese from one IPG to another during IEF by traversing buffer-filled porous bridges between the serial IPGs. A variety of materials can function as bridges, including paper, polyacrylamide gels or even IPGs. The quality of two-dimensional (2-D) protein patterns is not apparently worse than that generated by conventional IEF using the same individual IPGs. A major advantage of this method is that sample is consumed efficiently, without the requirement for preliminary steps, such as chamber IEF. This advantage is pronounced when working with extremely limited sources of samples, such as with clinical biopsies or cellular subfractions. The present study was limited by the commercial availability of suitable pH gradients. Proteomics analyses could be further improved if commercial vendors would manufacture IPGs with suitable pH ranges to achieve high resolution (approximately 100 cm) IEF separation of proteins in one electrophoretic step over the pH range 2-12. 相似文献
9.
The intraerythrocytic stage of Plasmodium falciparum alters the characteristics of its host cell by exporting selected plasmodial proteins. Although it is clear that the physicochemical and immunobiological properties of the host cell are modulated during parasite development, the involved plasmodial proteins and their mode of action are not completely known. Using cetyltrimethylammonium bromide (CTAB) or benzyldimethyl-n-hexadecylammonium chloride (16-BAC) for the first dimension and SDS for the second dimension, we separated proteins from membranes of human erythrocytes and of erythrocytes infected with the malaria parasite P. falciparum. Protein spots were analyzed by MALDI-TOF/TOF MS and annotated in respective 2D master gels. By using the alternative 2D approach, characteristic host cell membrane proteins and, more importantly, membrane-associated and exported plasmodial proteins were identified that might play a role in parasite-induced host cell modulation. 相似文献
10.
A two-dimensional proteome map of Shigella flexneri 总被引:14,自引:0,他引:14
Liao X Ying T Wang H Wang J Shi Z Feng E Wei K Wang Y Zhang X Huang L Su G Huang P 《Electrophoresis》2003,24(16):2864-2882
Shigella flexneri is a Gram-negative facultatively intracellular pathogen responsible for bacillary dysentery in humans. In this study, extracellular proteins from the culture medium and whole cell proteins in cellular extracts of S. flexneri 2a strain 2457T were examined by two-dimensional (2-D) gel electrophoresis using immobilized pH gradient (IPG) technology. Proteins were identified by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with Mascot search program. In total, among the 488 proteins spots processed, 388 proteins were identified. The identified proteins represented 169 genes. By comparing results of Mascot search against databases of Escherichia coli and genomes of S. flexneri 2a, one S. flexneri-specific protein was identified and one possible gap was found in 2457T genome sequences. Although this proteome map is still incomplete, it is already a useful reference for future studies involving pathogenicity, vaccine development, design of novel antibacterial drugs, etc. Proteome maps and a table of all identified proteins are available on the internet at www.proteomics.com.cn. 相似文献
11.
Pirovani CP Carvalho HA Machado RC Gomes DS Alvim FC Pomella AW Gramacho KP Cascardo JC Pereira GA Micheli F 《Electrophoresis》2008,29(11):2391-2401
Preparation of high-quality proteins from cacao vegetative organs is difficult due to very high endogenous levels of polysaccharides and polyphenols. In order to establish a routine procedure for the application of proteomic and biochemical analysis to cacao tissues, three new protocols were developed; one for apoplastic washing fluid (AWF) extraction, and two for protein extraction--under denaturing and nondenaturing conditions. The first described method allows a quick and easy collection of AWF--using infiltration-centrifugation procedure--that is representative of its composition in intact leaves according to the smaller symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the nondenaturing protein extraction and subsequent enzymatic activity detection. It is expected that the protocols described here could help to develop high-level proteomic and biochemical studies in cacao also being applicable to other recalcitrant plant tissues. 相似文献
12.
In this study, ampholyte-free liquid-phase IEF (LIEF) was combined with narrow pH range 2-DE and SDS-PAGE RP-HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2-DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2-DE gels following LIEF prefractionation into a single virtual 2-DE gel. Furthermore, the pH 3-5 LIEF fraction and the unfractionated sample were separated by pH 3-6 2-DE and identified by MALDI-TOF/TOF MS, respectively. In parallel, the pH 3-5 LIEF fraction was also analyzed by SDS-PAGE RP-HPLC MS/MS. LIEF-2-DE and LIEF-HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low-abundance proteins and proteins with extreme physicochemical characteristics or post-translational modifications compared to conventional 2-DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large-scale datasets. It was observed that the combination of LIEF-2-DE and LIEF-HPLC was effective in promoting MS-based liver proteome profiling and could be applied on similar complex tissue samples. 相似文献
13.
Marta Martin‐Lorenzo Laura Gonzalez‐Calero Irene Zubiri Pedro J. Diaz‐Payno Aroa Sanz‐Maroto Maria Posada‐Ayala Alberto Ortiz Fernando Vivanco Gloria Alvarez‐Llamas 《Electrophoresis》2014,35(18):2634-2641
Urine is a source of potential markers of disease. In the context of renal disease, urine is particularly important as it may directly reflect kidney injury. Current markers of renal dysfunction lack both optimal specificity and sensitivity, and improved technologies and approaches are needed. There is no clear consensus about the best sample pretreatment procedure for 2DE analysis of the urine proteome. Sample pretreatment conditions spots resolution and detection sensitivity, critically. As a first goal, we exhaustively compared eight different sample cleaning and protein purification methodologies for 2DE analysis of urine from healthy individuals. Oasis® HLB cartridges allowed the detection of the highest number of low molecular weight proteins; while PD10 desalting columns resulted in the highest number of detected spots in the high molecular weight area. Sample pretreatment strategies were also explored in the context of proteinuria, a clinical condition often associated to renal damage. Testing of urine samples from 13 patients with hypertension or kidney disease and different levels of proteinuria identified Oasis® HLB cartridge purification in combination with albumin depletion by ProteoPrep kit as the best option for urine proteome profiling from patients with proteinuric (> 30 mg/L albumin in urine) renal disease. 相似文献
14.
The purpose of this study was to develop a protocol suitable for membrane protein extraction from limited starting material and to identify appropriate conditions for two-dimensional (2-D) gel electrophoresis. We used A549 cells, a human alveolar type II cell line, and evaluated three protein extraction methods based on different separation principles, namely protein solubility, detergent-based and density-based organelle separation. Detergent-based extraction achieved the highest yield with 14.64% +/- 2.35 membrane proteins but sequential extraction with 7.35% +/- 0.78 yield and centrifugal extraction with 4.1% +/- 0.54 yield produced the purest fractionation of membrane proteins. Only the sequential and the detergent-based extraction proved suitable for small volumes of starting material. We identified annexin I + II, electron transfer flavoprotein beta-chain, H(+)-transporting ATP synthase, mitofilin and protein disulfide isomerase A3 as membrane and cytokeratin 8 + 18, actin and others as soluble proteins using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis and started to map the A549 cell proteome. Our data suggest that membrane proteins can be extracted efficiently from small samples using a simple sequential protein extraction method. They can be separated and identified successfully using optimized conditions in 2-D gel electrophoresis. The presented methods will be useful for further investigations of membrane proteins of alveolar and bronchial carcinomas. 相似文献
15.
As the new fluorescent stains such as SyproRuby and DeepPurple are getting widespread recognition for proteome analyses by the traditional 2-D gel method, it becomes important to test the feasibility of these stains with respect to staining reproducibility, protein quantitation, and compatibility of the stain with downstream MS. The binding of epicocconone, active ingredient of DeepPurple, to one of the primary cleavage sites of trypsin (lysine residue) raises the possibility of incomplete cleavage and interference with PMF. However, the current study tests and concludes that the DeepPurple stain can result in increased peptide recovery compared to SyproRuby stain and can improve MS-based identification of lower intensity proteins spots. 相似文献
16.
We investigated the protein profiles of variously aged rat astrocytes in response to oxidative stress. After H2O2-exposure of cells at 100 microM for 30 min, the relative intensity of ten protein spots changed on two-dimensional (2-D) gels compared with control gels after silver staining. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis after in-gel digestion revealed that six of these spots corresponded to three kinds of proteins, each of which was composed of a protein and its modified form with a different isoelectric point (pI). These three proteins were identified as peroxiredoxins (PRDXs) II and III, and calpactin I light chain (p11). H2O2-exposure increased the intensity of the spot with lower pI and simultaneously decreased that of the spot with higher pI for both PRDXs II and III. In addition, the expression of annexin VII, S-adenosyl-L-homocysteine hydrolase, elongation factor II fragment (EF-II), and adenosine deaminase was increased by H2O2-exposure in astrocytes from variously aged rats. Using the Pro-Q Diamond staining, heat shock protein 60 kDa (Hsp 60) and alpha-tubulin were observed to be phosphorylated upon H2O2-exposure. While phosphorylation of alpha-tubulin was correlated positively with age, the changes in abundance of ten protein spots as described above were independent of age. These results suggest that aging does not suppress the responses aimed at limiting injury and promoting repair brought about by severe oxidative stress, and might affect cell dynamics including the formation of microtubules. 相似文献
17.
In a previous publication a large series of proteins were identified in fetal human brain by the use of two-dimensional electrophoresis (2-DE) with subsequent matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and MALDI-tandem time-of-flight (TOF/TOF) analysis. Further identification of many more different spots by traditional 2-DE without additional step such as narrow immobilized ph gradient (IPG) strips or prefractionation seems unlikely and we therefore decided to separate extracted brain proteins by ion-exchange chromatography using a TSK gel DEAE-5PW column followed by 2-DE of individual fractions and analysis by MALDI-TOF/TOF with LIFT technology in fetal brain of the early second trimester. About 1880 protein spots corresponding to 543 different gene products were identified. These proteins included housekeeping, signaling, cytoskeletal, metabolic, antioxidant, and neuron/synaptosomal specific proteins. Among these, 314 gene products (314/543, 57.8%), which have never been detected in traditional 2-DE of human fetal brain, were observed by this method. This updated map of fetal brain proteins may serve as data base and reference map for fetal brain proteins, and the methodology applied may be used as a valuable analytical tool for the basis of protein expressional studies in health and disease. 相似文献
18.
The amniotic fluid cell proteome 总被引:2,自引:0,他引:2
Proteomic analysis of amniotic fluid cells may lead to the discovery of novel markers for embryonic abnormalities. A two-dimensional database for proteins of normal human amniotic fluid cells was constructed. The amniotic fluid cell extract was analyzed by two-dimensional gel electrophoresis and the proteins were identified by matrix-assisted laser desorption ionisation-time of flight-mass spectrometry. The database comprises 432 different gene products, which are in the majority enzymes, structural proteins, heat shock proteins, and proteins related to signal transduction. The obtained data show that the amniotic fluid population maybe either heterogeneous, originating from different fetal compartments and embryo tissues or is still pluripotent. Many proteins which are known to belong to certain cell types were found in the amnion cell fluid. This indicates that some types of fetal cells are already differentiated at the time of amniocentesis (about the 16(th) week of gestation). Moreover, the finding of proteins highly expressed in embryonic stem cells suggests that amniotic fluid could be used as a cell pool for transplantation therapy. 相似文献
19.
An important consideration in the measurement of quantitative changes in protein expression is the consistency of the observations for a given technique as well as the reproducibility of the experiment. A quantitative assessment of the technical and biological variability is crucial to avoid erroneous inferences and conclusions. Two methods for measuring quantitative changes in protein expression are two-dimensional electrophoresis (2-DE) and shotgun proteomics of isobaric-tagged samples using iTRAQ reagents. An assessment of changes in Escherichia coli protein expression in response to rhsA induction demonstrates that half of the quantified protein expression ratios have a coefficent of variation (CV) less than 0.31 using 2-DE and less than 0.24 using isobaric tags; whereas 95% of the quantified protein expression ratios have a CV less than 0.81 using 2-DE and less than 0.53 using isobaric tags. The selective removal of outlier data points from the shotgun method using Grubb's and Rosner's statistical outlier tests improves the consistency of the quantitation data obtained. 相似文献
20.
Mamone G Caira S Garro G Nicolai A Ferranti P Picariello G Malorni A Chianese L Addeo F 《Electrophoresis》2003,24(16):2824-2837
We report a fast and easy-to-use procedure that combines polyacrylamide gel electrophoresis with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF) and nanoelectrospray-tandem mass spectrometry (nES-MS/MS) analysis for the identification of casein components and defined phosphorylated sites. This methodology ensured identification of more than 30 phosphorylated proteins, five beta-, fifteen alpha(s1)-, ten alpha(s2)-, and four kappa-casein (CN) components, including nonallelic, differently phosphorylated, and glycosylated forms. The sugar motif covalently bound to kappa-CN was identified as chains, trisaccharide GalNAc, Gal, NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc. Also identified was a biantennary chain made up of both chains of trisaccharide 1GalNAc, 1Gal, 1NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc moiety on a single kappa-CN component. The phosphate group on site Ser12 of tryptic peptide 8-22 of most phosphorylated alpha(s1)-CN (11 phosphate groups) was localized and the oligosaccharide sequence of the main tryptic glycopeptides of two kappa-CN components was determined by means of MS/MS analysis. 相似文献