Two-dimensional fluorescence difference gel electrophoresis analysis of the urine proteome in human diabetic nephropathy

Urinary proteins may provide clues regarding pathogenesis of kidney disease as well as providing markers of disease activity. We employed two-dimensional differential in-gel electrophoretic analysis (2-D DIGE) to assess multiple urine samples in patients with diabetic nephropathy. Patient samples were collected as timed overnight collections. All the patients had longstanding diabetes, impaired renal function, and overt proteinuria. Control and patient urinary protein were analyzed by 2-D DIGE and DeCyder analysis. Ninety-nine spots were significantly regulated in the urine proteome of the diabetic samples, with 63 up- and 36 down-regulated. One spot corresponding to a pI 5-6 and a molecular weight between 45 and 66 kDa was consistently up-regulated by 19-fold across individuals in the diabetic group. Surface-enhanced laser desorption/ionization-time of flight analysis of in-gel tryptic digest of this spot identified this protein as alpha 1 antitrypsin (AAT). ELISA of urine samples from a separate group of patients and controls confirmed a marked increase of AAT in diabetic patients. Immunostaining of human diabetic kidneys revealed up-regulation of AAT in areas of renal fibrosis. In conclusion, we developed a method to analyze numerous urine samples from patients and allowed for detection and identification of regulated urine protein spots.     

Two-dimensional differential in gel electrophoresis (2D-DIGE) analysis of grape berry proteome during postharvest withering

The practice of postharvest withering is commonly used to correct quality traits and sugar concentration of high quality wines. To date, changes in the metabolome during the berry maturation process have been well documented; however, the biological events which occur at the protein level have yet to be fully investigated. To gain insight into the postharvest withering process, we studied the protein expression profiles of grape (Corvina variety) berry development focusing on withering utilizing a two-dimensional differential in gel electrophoresis (2D-DIGE) proteomics approach. Comparative analysis revealed changes in the abundance of numerous soluble proteins during the maturation and withering processes. On a total of 870 detected spots, 90 proteins were differentially expressed during berry ripening/withering and 72 were identified by MS/MS analysis. The majority of these proteins were related to stress and defense activity (30%), energy and primary metabolism (25%), cytoskeleton remodelling (7%), and secondary metabolism (5%). Moreover, this study demonstrates an active modulation of metabolic pathways throughout the slow dehydration process, including de novo protein synthesis in response to the stress condition and further evolution of physiological processes originated during ripening. These data represent an important insight into the withering process in terms of both Vitis germplasm characterization and knowledge which can assist quality improvement.     

Two-dimensional electrophoresis database of Listeria monocytogenes EGDe proteome and proteomic analysis of mid-log and stationary growth phase cells

Listeria monocytogenes is the causative agent of listeriosis, one of the most significant foodborne diseases in industrialized countries. The complete genome of the L. monocytogenes EGDe strain, belonging to the serogroup 1/2a, has been sequenced and is comprised of 2853 open reading frames. The objective of the current study was to construct a two-dimensional (2-D) database of the proteome of this strain. The soluble protein fractions of the microorganism were recovered either in the mid-log or in the stationary phase of growth at 37 degrees C. These fractions were analyzed by 2-D electrophoresis (2-DE), using immobilized pH gradient strips of various pH values (3-10, 3-6, and 5-8) for the first-dimensional separations and 12.5% acrylamide gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 201 protein spots corresponding to 126 different proteins were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The 2-DE maps presented here provide a first basis for further investigations of protein expression in L. monocytogenes. In this way, the comparison of proteome between cells in the exponential or stationary phase of growth at 37 degrees C allowed us to characterize 161 variations in protein spot intensity, of which 38 were identified. Among the differentially expressed proteins were ribosomal proteins (RpsF, RplJ, and RpmE), proteins involved in cellular metabolism (GlpD, PdhD, Pgm, Lmo1372, Lmo2696, and Lmo2743) or in stress adaptation (GroES and ferritin), a fructose-specific phosphotransferase enzyme IIB (Lmo0399) and different post-translational modified forms of listeriolysin (LLO).     

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.     

Two-dimensional strandness-dependent electrophoresis

Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acids in complex samples according to strandness, conformation and length. Under the non-denaturing conditions of the first electrophoretic step, single-stranded DNA, double-stranded DNA and RNA.DNA hybrids of similar length migrate at different rates. The second electrophoretic step is performed under denaturing conditions (7 mol l(-1) urea, 55 degrees C) so that all the molecules are single-stranded and separate according to length only. 2D-SDE is useful for revealing important characteristics of complex nucleic acid samples in manipulations such as amplification, renaturation, cDNA synthesis and microarray hybridization. It can also be used to identify mispaired, nicked or damaged fragments in double-stranded DNA. The protocol takes approximately 2 h and requires only basic skills, equipment and reagents.     

蛋白质组学研究中的双向电泳技术

蛋白质组学研究已经成为后基因组时代的研究热点,其两大支柱是双向凝胶电泳技术和生物质谱技术。尽管双向电泳技术近几年已经取得了突破性进展,是当前蛋白质分离的最常用技术,但其本身还有一些难以克服的问题。随着质谱技术的快速发展,双向电泳逐渐成为蛋白质组学研究的瓶颈。本综述双向电泳主要技术步骤的现状、存在问题及其改进方向。     

Two-dimensional gel electrophoresis characterization of the mouse leukocyte proteome, using a tri-reagent for protein extraction

The use of "tri-reagents" allows the concomitant isolation of DNA, RNA, and proteins for a complete molecular characterization of biological samples. The aim of the current study was to perform the comparative evaluation of two-dimensional gel proteomes isolated with or without a tri-reagent protein extraction step before dissolving the samples in the first-dimension electrophoresis buffer. We conclude that the use of tri-reagents increases the amount of protein extracted from the sample. Furthermore, an average of 301 +/- 3.6 spots were found in gels from both sample preparation methods, whereas 71.7 +/- 8.1 and 49.7 +/- 2.3 spots were uniquely seen in tri-reagent and non-tri-reagent samples, respectively.     

Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis

The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique. Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures. Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity. Whereas mitochondrial preparations isolated by means of differential centrifugation include a considerable degree of non-mitochondrial proteins (16%), this contamination could be effectually removed by the inclusion of a ZE-FFE purification step (2%). This higher degree of purity led to the identification of many more proteins from ZE-FFE purified mitochondrial protein extracts (n = 129), compared to mitochondrial protein extracts isolated by differential centrifugation (n = 80). Moreover, a marked decrease of degraded proteins was found in the ZE-FFE purified mitochondrial protein extracts. It is noteworthy that even at a low 2-DE resolution level, a four-fold higher number (17 versus 4) of presumably low abundance proteins could be identified in the ZE-FFE purified mitochondrial protein extracts. Therefore these results represent a feasible approach for an in-depth proteome analysis of mitochondria and possibly other organelles.     

Two-dimensional difference gel electrophoresis

Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as 'spots' with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With the appropriate imaging system, DIGE is capable of reliably detecting as little as 0.5 fmol of protein, and protein differences down to +/- 15%, over a >10,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2-3 d to complete.     

Two-dimensional electrophoresis computerized processing

This paper describes various methods suitable for implementation of two-dimensional processing software. The different steps leading to a complete processing are described, from the digitalization of the image to the processing of the resulting data. The characteristics of a convenient digitalization system are discussed. The different software devoted to spot detection is reviewed with respect to the presence or otherwise of a spot model and its characteristics. The major techniques for gel matching are compared as are designs for database structures suitable for tabulation of measurements. Finally, the need for a sophisticated system of data processing is stressed and its main requirements are described.     

Two-dimensional fluorescence difference gel electrophoresis analysis of spermatogenesis in the rat

The molecular mechanisms underlying normal and pathological spermatogenesis remain poorly understood. We compared protein concentrations in different germ cell types to identify those proteins specifically or preferentially expressed at each stage of rat spermatogenesis. Crude cytosolic protein extracts and reversed-phase HPLC prefractionated cytosolic extracts from spermatogonia, pachytene spermatocytes, and early spermatids were subjected to two-dimensional difference gel electrophoresis (2-D DIGE). By comparing gels and carrying out statistical analyses, we were able to identify 1274 protein spots with relative abundances differing significantly between the three cell types. We found that 265 of these spots displaying highly differential expression (ratio > or = 2.5 between two cell types), identified by mass fingerprinting, corresponded to 123 nonredundant proteins. The proteins clustered into three clades, corresponding to mitotic, meiotic, and post-meiotic cell types. The differentially expressed proteins identified by 2-D DIGE were confirmed and validated by western blotting and immunohistochemistry, in the few cases in which antibodies were available. 2-D DIGE appears a relevant proteomics approach for studying rat germ cell differentiation, allowing the establishment of the precise expression profiles for a relatively large number of proteins during normal spermatogenesis.     

Measuring gene expression by quantitative proteome analysis

Proteome analysis is most commonly accomplished by the combination of two-dimensional gel electrophoresis for protein separation, visualization, and quantification and mass spectrometry for protein identification. Over the past year, exceptional progress has been made towards developing a new technology base for the precise quantification and identification of proteins in complex mixtures, that is, quantitative proteomics.     

Two-dimensional gel electrophoresis in bacterial proteomics

Two-dimensional gel electrophoresis (2-DE) is a gel-based technique widely used for analyzing the protein composition of biological samples. It is capable of resolving complex mixtures containing more than a thousand protein components into individual protein spots through the coupling of two orthogonal biophysical separation techniques: isoelectric focusing (first dimension) and polyacrylamide gel electrophoresis (second dimension). 2-DE is ideally suited for analyzing the entire expressed protein complement of a bacterial cell: its proteome. Its relative simplicity and good reproducibility have led to 2-DE being widely used for exploring proteomics within a wide range of environmental and medically-relevant bacteria. Here we give a broad overview of the basic principles and historical development of gel-based proteomics, and how this powerful approach can be applied for studying bacterial biology and physiology. We highlight specific 2-DE applications that can be used to analyze when, where and how much proteins are expressed. The links between proteomics, genomics and mass spectrometry are discussed. We explore how proteomics involving tandem mass spectrometry can be used to analyze (post-translational) protein modifications or to identify proteins of unknown origin by de novo peptide sequencing. The use of proteome fractionation techniques and non-gel-based proteomic approaches are also discussed. We highlight how the analysis of proteins secreted by bacterial cells (secretomes or exoproteomes) can be used to study infection processes or the immune response. This review is aimed at non-specialists who wish to gain a concise, comprehensive and contemporary overview of the nature and applications of bacterial proteomics.     

Two-dimensional electrophoresis of small-molecular-weight proteins

We have developed a procedure for two-dimensional separation of small-molecular-weight (9000–30,000), acidic (pI 4–6) proteins that allows the use of strips cut from horizontal isoelectric-focusing slab gels for the first dimension, and discontinuous gels containing sodium dodecyl sulfate and high concentrations of urea in the second dimension. This technique facilitates the screening of large numbers of samples and the evaluation of electrofocusing artifacts. We emphasize measures to prevent major problems encountered in the use of this technique, particularly those caused by diffusion and aggregation. We also describe an extension of the method which allows the two-dimensional comparison of many samples in a selected narrow pH zone of interest.     

Two-dimensional gel proteome reference map of blood monocytes

Background:  

Blood monocytes play a central role in regulating host inflammatory processes through chemotaxis, phagocytosis, and cytokine production. However, the molecular details underlying these diverse functions are not completely understood. Understanding the proteomes of blood monocytes will provide new insights into their biological role in health and diseases.     

Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae

We have devised an approach for analyzing shotgun proteomics datasets based on the normalized spectral abundance factor that can be used for quantitative proteomics analysis. Three biological replicates of samples enriched for plasma membranes were isolated from S. cerevisiae grown in 14N-rich media and 15N-minimal media and analyzed via quantitative multidimensional protein identification technology. The natural log transformation of NSAF values from S. cerevisiae cells grown in 14N YPD media and 15N-minimal media had a normal distribution. The t-test analysis demonstrated 221 of 1316 proteins were significantly overexpressed in one or the other growth conditions with a p value <0.05. Notably, amino acid transporters were among the 14 membrane proteins that were significantly upregulated in cells grown in minimal media, and we functionally validated these increases in protein expression with radioisotope uptake assays for selected proteins.