High MS-compatibility of silver nitrate-stained protein spots from 2-DE gels using ZipPlates and AnchorChips for successful protein identification

The availability of easy-to-handle, sensitive, and cost-effective protein staining protocols for 2-DE, in conjunction with a high compatibility for subsequent MS analysis, is still a prerequisite for successful proteome research. In this article we describe a quick and easy-to-use methodological protocol based on sensitive, homogeneous, and MS-compatible silver nitrate protein staining, in combination with an in-gel digestion, employing the Millipore 96-well ZipPlate system for peptide preparation. The improved quality and MS compatibility of the generated protein digests, as compared to the otherwise weakly MS-compatible silver nitrate staining, were evaluated on real tissue samples by analyzing 192 Coomassie-stained protein spots against their counterparts from a silver-stained 2-DE gel. Furthermore, the applicability of the experimental setup was evaluated and demonstrated by the analysis of a large-scale MALDI-TOF MS experiment, in which we analyzed an additional ~1000 protein spots from 2-DE gels from mouse liver and mouse brain tissue.     

Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography

We describe an approach for fractionating complex protein samples prior to two-dimensional gel electrophoresis using reversed-phase high-performance liquid chromatography. Whole lysates of cells and tissue were prefractionated by reversed-phase chromatography and elution with a five-step gradient of increasing acetonitrile concentrations. The proteins obtained at each step were subsequently separated by high-resolution two-dimensional gel electrophoresis (2-DE). The reproducibility of this prefractionation technique proved to be optimal for comparing 2-DE gels from two different cell states. In addition, this method is suitable for enriching low-abundance proteins barely detectable by silver staining to amounts that can be detected by Coomassie blue and further analyzed by mass spectrometry.     

A proteome database of human primary T helper cells

We have established the first public database of human primary T helper cell proteome using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry. For the database, CD4+ human T cells were activated with anti-CD3+anti-CD28 antibodies and metabolically labeled with [35S]methionine for 24 h. Cells were lysed and proteins were separated by 2-DE. About 1500 protein spots were detected in the resulting 2-DE gels with silver staining, and 2000 spots with autoradiography. We have identified 91 proteins from the 2-DE gels using peptide mass fingerprinting, and annotated them to our database. The identified proteins are also linked to SWISS-PROTand NCBI protein databases. Our database is available via the Internet at http://www3.btk.utu.fi:8080/Genomics/Proteomics/Database.     

Reference map of soluble proteins from Streptococcus thermophilus by two-dimensional electrophoresis

Streptococcus thermophilus is a lactic acid bacterium widely used for the production of fermented dairy products. The two-dimensional electrophoresis (2-DE) protein profile was obtained from three independent analyses of 2-DE gels of soluble proteins of the strain PB18. About 270 spots were detected by silver staining and the average molecular weight and isoelectric point of each protein spot were calculated to be 41 600 and 5.2, respectively. Twelve proteins were purified by chromatographic techniques because their concentration was too low for direct sequencing from blots. Eleven were located in the PB18 2-DE profile after silver staining. These preliminary results contribute to the setting up of a two-dimensional image (or reference map) of the proteins from S. thermophilus in order to identify and compare strains of various origin or to follow metabolic process such as stress. Bidimensional autoradiographs of two strains (PB18 and ST105) of S. thermophilus grown in exponential phase at 42 degrees C with [35S]methionine were compared with an image analysis system. Among the eleven located proteins in the 2-DE silver-stained profile, nine were found in PB18 and eight in ST105 autoradiographs. One protein was specific to PB18. The eight proteins could play the role of internal 2-D PAGE markers of p/ and Mr for S. thermophilus.     

适于双向电泳分析的苹果叶片蛋白质提取方法

为了探索适用于双向电泳(2-DE)分析的苹果叶片蛋白质提取方法,比较了三氯乙酸(TCA)/丙酮沉淀法、二硫苏糖醇(DTT)/丙酮法、Tris-HCl提取法和改良的Tris-HCl提取法等4种蛋白质提取方法。以7 cm、pH 3～10的线性固相pH梯度(immobilized pH gradient,IPG)胶条作为第一向电泳,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)(12.5%的分离胶)作为第二向电泳,对提取物进行2-DE分离,采用银染显色。结果表明,上述4种方法在2-DE图谱上分别得到140,215,181和616个蛋白质点。其中以改良的Tris-HCl提取法得到的蛋白质点数最多,且背景清晰、图谱上没有明显的横纵条纹。为了进一步验证改良的Tris-HCl提取法的有效性,用18 cm、pH 3～10的线性IPG胶条和12.5%的分离胶对提取的苹果叶片蛋白质进行2-DE分离,考马斯亮蓝R-250染色,共检测到455个蛋白质点,其相对分子质量主要分布在14000～66000范围内,图谱背景清晰,再次证明应用该方法制备的样品适用于双向电泳分析,可用于苹果叶片的蛋白质组学分析。     

Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain

The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one- and two-dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein-specific, ranging from 0.5 to 5 ng. The linearity of the relationship between protein level and SYPRO Ruby staining intensity also depended on the individual protein, with observed linear dynamic ranges of 200-, 500-, and, 1000-fold for proteins analyzed by SDS-PAGE. SYPRO Ruby protein gel stain was also evaluated in two-dimensional electrophoretic (2-DE) analysis of Escherichia coli proteins. The experiment involved analysis of replicates of the same sample as well as dilution of the sample from 0.5 to 50 nug total protein across gels. In addition to validating the 2-DE system itself, the experiment was used to evaluate three different image analysis programs: Z3 (Compugen), Progenesis (Nonlinear Dynamics), and PDQuest (Bio-Rad). In each program, we analyzed the 2-DE images with respect to sensitivity and reproducibility of overall protein spot detection, as well as linearity of response for 20 representative proteins of different molecular weights and pI. Across all three programs, coefficients of variation (CV) in total number of spots detected among replicate gels ranged from 4 to 11%. For the 20 representative proteins, spot quantitation was also comparable with CVs for gel-to-gel reproducibility ranging from 3 to 33%. Using Progenesis and PDQuest, a 1000-fold linear dynamic range of SYPRO Ruby was demonstrated with a single known protein. These two programs were more suitable than Z3 for examining individual protein spot quantity across a series of gels and gave comparable results.     

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

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

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

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

Establishment of “one-piece” large-gel 2-DE for high-resolution analysis of small amounts of sample using difference gel electrophoresis saturation labelling

Large-gel two-dimensional gel electrophoresis (2-DE) is the method of choice for high-resolution proteome analysis of complex protein mixtures. Until now, however, the advantages of large 2-DE in combination with multiplexed fluorescence dye protein labelling has been complicated by the separate handling and analysis of the second-dimension gels. Therefore, we adapted the large 2-DE procedure allowing us to run “one-piece” large 2-DE gels (40 cm × 30 cm) in the second dimension for high resolution proteome analysis. Here, we show that in combination with fluorescence dye protein saturation labelling “one-piece” large 2-DE enables analysis of small amounts of sample (3 μg protein) for high-resolution proteome analysis.     

Dialysis-assisted two-dimensional gel electrophoresis

2-DE is an important tool in proteomics research. However, intrinsic gel-to-gel variability of 2-DE often masks the biological differences between the samples and compromises quantitative comparison of protein expression levels. Here, we describe a modification of 2-DE that results in improved matching and quantification of proteins. This was accomplished by performing IEF of two samples in two IPG strips separated by a dialysis membrane. After IEF running, the strips were separated and the SDS-PAGE dimension was accomplished on two individual gels. After gel staining with CBB, ImageMaster 2D Platinum software (Amersham) was used for spot detection and quantification. Analysis of protein extracts from C2C12 myoblasts by this method resulted in 99% spot-matching efficiency and CV in stain intensity (% volume) was less than 0.5 for 98% of spots. We conclude that this technique, called dialysis-assisted gel electrophoresis, gives superior spot matching and quantitative reproducibility compared to IEF conducted on separate strips.     

Quantitation of human leukocyte proteins after silver staining: a study with two-dimensional electrophoresis.

The quantitative attributes of human leukocyte proteins detected by silver staining two-dimensional electrophoresis (2-DE) gels were studied by using computer-assisted data analysis. Experiments included (a) analysis of replicate patterns of the same sample, (b) analysis of different dilutions of the same sample, and (c) analysis of samples from different individuals. Over 200 proteins were observed to have coefficients of variation (CV) less than or equal to 15% when data from replicate patterns were analyzed. In contrast, 8 proteins had CV values of less than or equal to 15% when data from different samples were analyzed. The dilution experiment showed that a majority of the proteins detected with some consistency (i.e., observed in at least 80% of the patterns) have a linear relationship between the amount of protein loaded onto a 2-DE gel and the spot volume in the final 2-DE pattern. The slope of the curves and the deviation from linearity were found to be quite protein-specific. These results indicate that optimization of sample purity and minimization of staining protocol variables are required to limit the background quantitative variability between and within 2-DE runs to a level that will allow detection of quantitative changes indicative of biological responses.     

Fluorogenic Ag+–Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining

Silver staining, which exploits the special bioaffinity and the chromogenic reduction of silver ions, is an indispensable visualization method in biology. It is a most popular method for in‐gel protein detection. However, it is limited by run‐to‐run variability, background staining, inability for protein quantification, and limited compatibility with mass spectroscopic (MS) analysis; limitations that are largely attributed to the tricky chromogenic visualization. Herein, we reported a novel water‐soluble fluorogenic Ag⁺ probe, the sensing mechanism of which is based on an aggregation‐induced emission (AIE) process driven by tetrazolate‐Ag⁺ interactions. The fluorogenic sensing can substitute the chromogenic reaction, leading to a new fluorescence silver staining method. This new staining method offers sensitive detection of total proteins in polyacrylamide gels with a broad linear dynamic range and robust operations that rival the silver nitrate stain and the best fluorescent stains.     

Two-dimensional gel electrophoresis of platelet polypeptides with immobilized pH gradients in capillary tubes

Two-dimensional electrophoresis (2-DE) with immobilized pH gradient (IPG) gels in capillary tubes was used in the first-dimensional isoelectric focusing (IEF) for the separation of human platelet polypeptides. Two types of IPG tube gels, pH ranges 4-8 and 7-10, containing 8 M urea, 1% Nonidet P-40 and 0.1% pH 3.5-10 Ampholine carrier ampholytes (CA) were prepared by a simple method not requiring special equipment. The addition of CA to both gel and sample solutions was essential in the tube gel IPG system. Proteins were visualized by a modification of Wray's silver-staining technique. The degree of resolution and the number of spots observed on an IPG 2-DE gel with pH 4-8 were comparable with those obtained with O'Farrell's high-resolution 2-DE. Approximately 200 basic polypeptides, which are difficult to separate by conventional CA-based IEF 2-DE or the non-equilibrium pH gradient system, were well resolved by 2-DE with a pH 7-10 IPG tube gel in the first-dimension. The gel patterns with either pH gradient 4-8 or 7-10 were highly reproducible among gels prepared and run simultaneously. These results demonstrated the potential and usefulness of the 2-DE system with IPG gels in capillary tubes.     

Improved dynamic range of protein quantification in silver‐stained gels by modelling gel images over time

Silver staining is a commonly used protein stain to visualise proteins separated by 2‐DE. Despite this, the technique suffers from a limited dynamic range, making the simultaneous quantification of high‐ and low‐abundant proteins difficult. In this paper we take advantage of the fact that silver staining is not an end‐point stain by photographing the gels during development. This procedure provides information about the change in measured absorbance for each pixel in the protein spots on the gel. The maximum rate of change was found to be correlated with the amount of applied protein, providing a new way of estimating protein amount in 2‐DE gels. We observed an improvement in the dynamic range of silver staining by up to two orders of magnitude.     

Large-scale muLC-MS/MS for silver- and Coomassie blue-stained polyacrylamide gels

2-DE combined with LC-MS/MS has become a routine, reliable protein separation and identification technology for proteome analysis. The demand for large-scale protein identifications after 2-DE separation requires a sensitive and high-throughput LC-MS/MS method. In this report, a simple, splitless, fully automated capillary LC-MS/MS system was described for the large-scale identification of proteins from gels stained with either silver or CBB. The gel samples were digested and peptides were extracted using an in-gel digestion workstation. The peptides were automatically introduced into a capillary column by an autosampler connected to an HPLC pump. A nanoLC pump was then used to deliver the gradient and elute the peptides from the capillary column directly into an LCQ IT mass spectrometer. Neither a peptide trapping setting nor a flow split is needed in this simple setup. The collected MS/MS spectra were then automatically searched by SEQUEST, and filtered and organized by DTASelect. Hundreds of silver-stained or CBB-stained Shewanella oneidensis, Geobacter sulfurreducens, and Geobacter metallireducens proteins separated by denaturing or nondenaturing 2-DE were digested and routinely analyzed using this fully automated muLC-MS/MS system. High peptide hits and sequence coverage were achieved for most CBB-stained gel spots. About 75% of the spots were found to contain multiple proteins. Although silver staining is not commonly thought to be optimal for MS analysis, protein identifications were successfully obtained from silver-stained 2-DE spots detected using methods with and without formaldehyde for protein fixation.     

Quantitative and qualitative measure of intralaboratory two-dimensional protein gel reproducibility and the effects of sample preparation, sample load, and image analysis

We investigate one approach to assess the quantitative variability in two-dimensional gel electrophoresis (2-DE) separations based on gel-to-gel variability, sample preparation variability, sample load differences, and the effect of automation on image analysis. We observe that 95% of spots present in three out of four replicate gels exhibit less than a 0.52 coefficient of variation (CV) in fluorescent stain intensity (% volume) for a single sample run on multiple gels. When four parallel sample preparations are performed, this value increases to 0.57. We do not observe any significant change in quantitative value for an increase or decrease in sample load of 30% when using appropriate image analysis variables. Increasing use of automation, while necessary in modern 2-DE experiments, does change the observed level of quantitative and qualitative variability among replicate gels. The number of spots that change qualitatively for a single sample run in parallel varies from a CV = 0.03 for fully manual analysis to CV = 0.20 for a fully automated analysis. We present a systematic method by which a single laboratory can measure gel-to-gel variability using only three gel runs.     

High reproducibility of large-gel two-dimensional electrophoresis

Two-dimensional gel electrophoresis (2-DE) facilitates the separation of thousands of proteins from highly complex protein mixtures and has become a central method in proteomics in recent years. In the present study, we examined the technical variability of large 2-DE gels with respect to sample preparation, electrophoresis procedure, data acquisition, and biological variation by analyzing a disease (Huntington's disease) and control state with a commercially available software package, PROTEOMWEAVER trade mark. Scatter plots and correlation coefficients were obtained to quantify both technical and biological variation. Even 2-DE gels run separately in both dimensions yielded correlation coefficients around 0.88 and deviations from the mean close to 20% for low-intensity spots. This indicates a high technical reproducibility of the 2-DE procedure developed in our laboratory. Variability within a biological condition was low and comparable to technical variation (at least 0.87). Two-dimensional (2-D) gels obtained from samples of different biological conditions (health vs. disease) achieved a variability similar to intracondition and technical variability. These findings highlight the importance of multiple gel and spot-by-spot comparisons to identify biological significant changes. Minor errors introduced by technical and biological variation allow a comparison of all gels within a study which facilitates the tackling of complex biological problems.     

Proteomic analysis of carbonylated proteins in two-dimensional gel electrophoresis using avidin-fluorescein affinity staining

A method for detecting carbonylated proteins in two-dimensional electrophoresis (2-DE) was developed using biotinylation and avidin-fluorescein isothiocyanate (FITC) affinity staining. The method was used to examine oxidatively modified proteins associated with oxidative stress. Carbonyl formation in proteins was first examined in a model system by subjecting bovine serum albumin (BSA) and ribonuclease A (RNase A) to metal-catalyzed oxidation (MCO). Carbonyl group formation was found to occur at multiple sites along with a small amount of polypeptide chain cleavage. In vivo studies were conducted in yeast cell cultures using 5 mM hydrogen peroxide to induce oxidative stress. Biotinylation of yeast protein was accomplished during extraction at 4 degrees C in a lysis buffer containing 5 mM biotin-hydrazide. Biotin-hydrazide forms a Schiff base with a carbonyl group on an oxidized protein that is subsequently reduced before electrophoresis. Proteins were separated by either 2-DE or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biotinylated species were detected using avidin-FITC affinity staining. Detection sensitivity with biotinylated proteins was five times higher than achieved by silver staining. The limit of detection with avidin-FITC staining approached 0.64 pmol of protein-associated carbonyls. Twenty carbonylated proteins were identified in the proteome of yeast following oxidative stress with hydrogen peroxide. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis of tryptic peptides was used to identify peptides extracted from gels. Aconitase, heat shock protein SSA1 and SSC1, pyruvate decarboxylase isozyme 1, pyruvate kinase 1, enolase 1 and 2, phosphoglycerate kinase, fructose-bisphosphate aldorase, and glyceraldehyde-3-phosphate dehydrogenase were among the major targets of oxidative stress.     

A human myocardial two-dimensional electrophoresis database: protein characterisation by microsequencing and immunoblotting.

This communication briefly describes how a human heart two-dimensional electrophoresis (2-DE) protein database is being established in our laboratory. The database contains more than 1500 polypeptides and approximately fifty proteins from 2-DE gels of human myocardial tissue have been characterised. Information about the proteins has been compiled including molecular weight (M(r)), isoelectric point (pI), sample spot (SSP) number, protein name, partial sequence, and antibody reacting with the protein. The first stage of this project involves the investigation of protein with pIs in the range pH 4-7. Future studies will employ immobilised pH gradient (IPG) gels as the first dimension of the 2-DE to examine basic proteins. The ultimate goal of this project is to establish a global picture of human heart protein expression in both normal and disease conditions.     

Proteomic analysis of immunostained,laser-capture microdissected brain samples

Proteomic analysis is often performed on homogenized preparations of whole tissues, which does not provide any information about relevant biochemical changes in specific cell types. Laser-capture microdissection (LCM) is a technique that is precise enough to dissect single cells within a tissue section. Phenotypically defined cells of interest may be visualized by immunostaining prior to microdissection. Previously published immunostaining protocols adapted to LCM require the use of very high antibody titers and very short incubation times. This raises the concern that low-abundance antigens would not be detected and that antisera would be rapidly depleted. In addition, protein recovery from samples was not evaluated in most of these studies. Here, we describe an optimized immunostaining method based on immunofluorescence. By comparing two-dimensional electrophoresis (2-DE) results obtained from immunostained LCM brain tissue samples to those obtained from unstained, manually dissected samples, we demonstrated that immunofluorescent staining gave comparable protein recovery and similar resolution of protein spots on 2-DE gels. Moreover, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis of selected spots from gels derived from control and immunostained LCM samples revealed that the immunostaining process had minimal effect on protein identification. LCM of immunofluorescently labeled tissue sections is a practical and powerful method to perform proteomic studies on specifically defined cell groups.