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.     

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。     

Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis

The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.     

Phosphoproteome profile of human liver Chang's cell based on 2-DE with fluorescence staining and MALDI-TOF/TOF-MS

Because reversible protein phosphorylation is central to biological regulation, many methods have been developed for the systematic parallel analysis of the phosphorylation status of large sets of proteins. To directly survey the extent of protein phosphorylation and the distribution of phosphoproteins in biological systems, we used a phosphoprotein staining method, Pro-Q Diamond dye, for the high-throughput identification of phosphoproteins. The specificity of the method was validated with protein standards and subsequently applied to an analysis of total protein from human liver Chang's cells. Proteins were separated by 2-DE, then sequentially stained with Pro-Q Diamond and Coomassie Blue G-250. After image analysis, the proteins in gel spots containing phosphoproteins were identified by MALDI-TOF/TOF-MS. A total of 269 phosphoproteins were identified, and 27 were known phosphoproteins in the SwissProt database. By comparing the relative volumes of the phosphoprotein map and the total protein map, the extent of protein phosphorylation was observed. The phosphoprotein staining method combined with 2-DE also detected polymorphisms of the phosphoproteins, and could distinguish highly abundant, but slightly phosphorylated proteins from less abundant, highly phosphorylated ones. We conclude that the phosphoprotein staining method can be used for global, quantitative phosphorylation detection.     

Substituting Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis buffers improves the resolution of focusing patterns

In a new area of postgenomics challenges, the optimization of protein identification has become a central goal in microbiochemistry. In this work, we demonstrate that the substitution of Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis (2-DE) buffers improves the focusing of whole proteins from Pseudomonas aeruginosa. This improvement of focusing concerns more particularly basic proteins. This enhancement may be attributed to a better transfer from the first to the second dimension, which probably highlights an increase in the solubility of proteins in the IPG strips. Hence, the use of an efficient tracking dye in the 2-DE buffers may enlarge protein recovery on proteome maps.     

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.     

Systematic comparison of technical details in CBB methods and development of a sensitive GAP stain for comparative proteomic analysis

Considering the importance of CBB staining in visualizing proteins in 2-DE gels, any improvement in the existing protocols with high sensitivity and good MS compatibility is of significant importance. In this study, we systematically evaluated the effects of different staining parameters on CBB methods by 1-DE and 2-DE, and demonstrated that G-250 was more suitable for visualizing low-abundant proteins as well as generating more spots than R-250, whereas R-250 had a superior capability for quick staining of high-abundant proteins. The staining produced by mixing G-250 and R-250 in different ratios showed similar sensitivity. Compared with acetic acid, phosphoric acid produced more protein spots. Ammonium-based stain demonstrated a superior sensitivity than the aluminum-based one. Based on these findings, a new protocol using CBB G-250, ammonium sulfate and phosphoric acid (GAP) was developed by incorporating the fixation, sensitization and staining procedures together. The comparison of GAP with other methods revealed that GAP generated more protein spots and had wider applications. The identification of 11 proteins demonstrated that GAP was not only compatible with MS but also obviously reduced in vitro protein modification, and thus could be a preferable protocol in the future proteomic analysis.     

Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250

An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.     

Study of Burkitt lymphoma cell line proteins by high resolution two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry

We paper describe a mass spectrometric approach generally applicable for the rapid identification and characterization of proteins isolated by two-dimensional gel electrophoresis (2-DE). The highly sensitive nanoflow-electrospray mass spectrometry employing a quadrupole-time of flight mass spectrometer was used for the direct identification of proteins from the peptide mixture generated from only one high resolution 2-DE gel without high performance liquid chromatography (HPLC) separation or Edman sequencing. Due to the high sensitivity and high mass accuracy of the instrument employed, this technique proved to be a powerful tool for the identification of proteins from femtomole amounts of materials. We applied the technique for the investigation of Burkitt lymphoma BL60 cell proteins. This cell line has been used as a model to assign apoptosis-associated proteins by subtractive analysis of normal and apoptotic cells. From the nuclear fraction of these cells, 36 protein spots were examined, from only one micropreparative Coomassie Brilliant Blue R-250 stained gel, after proteolytic digestion by matrix assisted laser desorption ionization (MALDI) and nanospray mass spectrometry (MS). In combination with database searches, of 33 proteins were successfully identified by nanospray-MS/MS-sequencing of up to eight peptides per protein. Three proteins were new proteins not listed in any of the available databases. Some of the identified proteins are known to be involved in apoptosis processes, the others were common proteins in the eukaryotic cell. The given technique and the protein data are the basis for construction of a database to compare normal and apoptosis-induced cells and, further, to enable fast screening of drug impact in apoptosis-associated processes.     

Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics

Two-dimensional gel electrophoresis (2-DE) is currently the best method for separating complex mixtures of proteins, and its use is gradually becoming more common in cardiac proteome analysis. A number of variations in basic 2-DE have emerged, but their usefulness in analyzing cardiac tissue has not been evaluated. The purpose of the present study was to systematically evaluate the capabilities and limitations of several 2-DE techniques for separating proteins from rat heart tissue. Immobilized pH gradient strips of various pH ranges, parameters of protein loading and staining, subcellular fractionation, and detection of phosphorylated proteins were studied. The results provide guidance for proteome analysis of cardiac and other tissues in terms of selection of the isoelectric point separating window for cardiac proteins, accurate quantitation of cardiac protein abundance, stabilization of technical variation, reduction of sample complexity, enrichment of low-abundant proteins, and detection of phosphorylated proteins.     

Cell surface proteins of Candida albicans: preparation of extracts and improved detection of proteins

We have reexamined the detection of the components in a beta-mercaptoethanol and ammonium carbonate buffer extract of surface proteins of Candida albicans and the effects of postextraction manipulation of the extract on recovery of extract components. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), preferential staining of some moieties was observed when bands detected by a commercial silver staining method or a Coomassie Brilliant Blue (CBB) staining method were compared. Additional protein bands that were either not detected or poorly detected by a single method alone were readily observed by a combined silver-CBB staining method. This method also detected alterations in the profile of extracted proteins from organisms grown in the presence of galactose or hemoglobin rather than glucose. Two-dimensional electrophoresis (2-DE) gel analysis by double stain showed better detection of several acidic and basic protein spots. Less than 10% of the extract as determined by a dye-binding assay was lost following either or both lyophilization and dialysis. These manipulations of the extract did not change the protein profile following SDS-PAGE as determined by the combined staining or Western blot analysis of a 70 kDa protein. These observations suggest that soluble cell wall proteins are not unusually sensitive to procedures routinely used in protein purification. In addition, these studies suggest that a modified staining method that combines both silver stain and CBB stain provides improved detection of cell wall proteins compared to either method alone.     

Increased sensitivity for Coomassie staining of sodium dodecyl sulfate-polyacrylamide gels using PhastSystem Development Unit

Staining of proteins in PhastGel gradient media with Coomassie Blue R 350 was considerably improved using a lower concentration of methanol (10% v/v) and 2% ammonium sulfate in the staining solution and 10% acetic acid for destaining. The detection limit in sodium dodecyl sulfate-polyacrylamide gels was lowered by a factor of 10 to about 2 ng per protein band. The Coomassie staining method was adapted to the newly developed silver staining procedure so that both can be used in parallel in PhastSystem.     

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.     

A plant dye from Lawsonia inermis for protein staining after polyacrylamide gel electrophoresis.

A reddish-brown dye was isolated from the leaves of Lawsonia inermis by extraction with calcium hydroxide (pH 11-12). A 3.6% crude extract in ethanol/water, 1:1 v/v, was used for direct staining, without fixation, of bovine serum albumin, casein and human serum proteins, following polyacrylamide gel electrophoresis in cylindrical gels. After staining for 30 min the gels were destained for 1/2-2 h with 7% acetic acid at 60 degrees C. Protein staining with Amido Black 10B and Coomassie Brilliant Blue R-250, according to standard protocols, required destaining for 24 h and more to obtain a comparably cleared background. Staining with the plant dye and Coomassie Brilliant Blue had a similar overall staining sensitivity but some minor components of human serum showed different staining characteristics with each of the two dyes. Staining with the plant dye excels over standard staining by speed and simplicity.     

Comparison of different staining methods for polyvinylidene difluoride membranes.

Several new staining methods for polyvinylidene difluoride membranes, including mercurochrome, silver and dimethylaminoazobenzene isothiocyanate staining were compared with Coomassie Brilliant Blue and gold staining. Of these, Coomassie was most versatile and completely compatible with ensuing microsequencing, immunostaining or other visualization methods, while gold and silver staining were more sensitive. Mercurochrome allows selective detection of sulfhydryl-containing proteins while dimethylaminoazobenzene isothiocyanate staining may allow quantitation of sequenceable protein.     

Improved detection of lymphocyte membrane proteins in purified form and as a crude mixture using native and denaturing polyacrylamide gel electrophoresis by optimisation of coomassie brilliant blue and silver staining.

Optimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol-acetic acid-formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.     

缺氧预处理诱导心肌细胞蛋白质组变化的初步研究

缺氧预处理(hypoxia preconditioning, HPC)可模拟缺血预处理(ischemic preconditioning, IPC)对缺血/再灌注心肌的保护作用, 涉及细胞内众多分子事件. 本工作旨在采用双向电泳和质谱分析等蛋白质组分析技术, 发现缺氧预处理后心肌细胞蛋白质整体表达上的变化, 初步分析其与缺氧预处理心肌保护作用的关系. 将原代培养的SD乳鼠心肌细胞分为2组(n＝6): (1)缺氧预处理组(HPC): 将细胞置缺氧仓内短暂缺氧20 min进行缺氧预处理(HPC), 制备心肌细胞蛋白提取物; (2)对照组(control): 细胞置于培养箱内持续常氧孵育至实验结束, 提取蛋白. 采用双向凝胶电泳和图像扫描, 经蛋白样本分离和考马斯亮蓝染色后比较分析, 选取3个差异表达蛋白点进行胶内酶切、肽质量指纹图谱分析和数据库检索. 双向电泳可分离约529±45个蛋白质, 点匹配率约为78%±7.5%. 18种蛋白质在HPC后发生明显表达差异, 其中12种蛋白质表达降低, 6种表达增高. 经质谱分析鉴定出的3种蛋白质分别为myosin light polypeptide 3, nucleoside diphosphate kinase (NDPK)和calreticulin (CRT). 缺氧预处理引起心肌细胞蛋白质组变化, 初步发现其中myosin light polypeptide 3表达下调、nucleoside diphosphate kinase和calreticulin表达增加, 可能通过调节心肌细胞的收缩性、激活G蛋白、调节细胞内Ca^2＋浓度而保护心肌. 本工作通过研究缺氧预处理延迟保护过程中心肌内源性蛋白表达水平的变化, 有助于从细胞水平探讨预处理延迟保护机制.     

Proteome database of unsensitized CD4 positive T lymphocytes in T cell receptor transgenic mice

We established a two-dimensional electrophoresis (2-DE) mapping database of splenic CD4 T cells prepared from I-A(d)-restricted ovalbumin (OVA)(323-339) specific T cell receptor (TCR) transgenic mice (OVA23-3). First we examined the purification of CD4 T cells and found that the high purity of cells produced more accurate protein maps. The first dimension utilized narrow-range immobilized pH gradients (IPGs), pH 4.0-5.0, pH 4.5-5.5, pH 5.0-6.0, and pH 5.5-6.7. Approximately 1300 spots were detected by silver staining. Detection was performed by in-gel tryptic digestion of the spots, matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) technology and database searches via the world wide web (WWW). We have so far identified 255 proteins on 2-DE gels of whole cell lysates. This is the first construction of a proteome database for murine unsensitized CD4 T lymphocytes. To examine this further, 2-DE mapping was utilized for splenic CD4 T cells from another TCR transgenic mouse strain (DO11.10 TCR transgenic mice). Mapping patterns were found to be almost identical to those from CD4 T cells from OVA23-3 mice. These results indicated that the 2-DE maps in this study could be used for mouse CD4 T cells to examine protein changes in cells given certain stimuli.     

Glucose/Ribitol Dehydrogenase and 16.9 kDa Class I Heat Shock Protein 1 as Novel Wheat Allergens in Baker’s Respiratory Allergy

Wheat allergens are responsible for symptoms in 60–70% of bakers with work-related allergy, and knowledge, at the molecular level, of this disorder is progressively accumulating. The aim of the present study is to investigate the panel of wheat IgE positivity in allergic Italian bakers, evaluating a possible contribution of novel wheat allergens included in the water/salt soluble fraction. The water/salt-soluble wheat flour proteins from the Italian wheat cultivar Bolero were separated by using 1-DE and 2-DE gel electrophoresis. IgE-binding proteins were detected using the pooled sera of 26 wheat allergic bakers by immunoblotting and directly recognized in Coomassie stained gel. After a preparative electrophoretic step, two enriched fractions were furtherly separated in 2-DE allowing for detection, by Coomassie, of three different proteins in the range of 21–27 kDa that were recognized by the pooled baker’s IgE. Recovered spots were analyzed by nanoHPLC Chip tandem mass spectrometry (MS/MS). The immunodetected spots in 2D were subjected to mass spectrometry (MS) analysis identifying two new allergenic proteins: a glucose/ribitol dehydrogenase and a 16.9 kDa class I heat shock protein 1. Mass spectrometer testing of flour proteins of the wheat cultivars utilized by allergic bakers improves the identification of until now unknown occupational wheat allergens.