Proteomic analysis of colonic crypts from normal, multiple intestinal neoplasia and p53-null mice: a comparison with colonic polyps

In order to observe cellular changes caused by mutation of the tumor suppressors, APC and p53, we have generated protein expression profiles of mouse colon epithelial cells using two-dimensional electrophoresis (2-DE). Crypts, polyps and stroma were isolated from normal, multiple intestinal neoplasia (MIN) and p53-null mice, each with a C57Black/6J background, and subjected to 2-DE in two separate pH ranges (pH 3-10 and pH 6-11). No significant differences in protein expression patterns were observed between the normal, MIN and p53-null colon epithelial crypts. However, 64 proteins from the MIN polyps showed a 2-fold or greater difference in intensity that was statistically significant as assessed by the Wilcoxon rank-sum test (p < or = 0.05). Of these, calreticulin, carbonic anhydrase I and a new member of the glutathione-S-transferase theta family of proteins have so far been identified using an in-gel digestion protocol coupled with reversed-phase high performance liquid chromatography (RP-HPLC) ion-trap mass spectrometry. In addition, 38 marker proteins have been identified in a continuing effort to generate a comprehensive 2-DE database of proteins expressed by mouse colon epithelial cells (these databases are available at http://www.ludwig.edu.au/jpsl/jpslhome. html).     

Dynamics of Arabidopsis thaliana soluble proteome in response to different nutrient culture conditions

In an effort to determine the best extraction procedure compatible with the high-reproducible 2-DE, different methods of soluble protein extraction from Arabidopsis cell culture suspensions grown in Gamborg B5 medium were tested. A reference 2-DE map was established for this soluble extract revealing 1184 spots. The most abundant protein spots were excised, trypsin-digested, and mass spectra obtained via MALDI-TOF and/or LC coupled to ESI-MS. Three hundred and thirty one proteins were identified and their functions were defined based on sequence comparisons and classified in different protein families. In order to analyze the impact of culture medium on the Arabidopsis proteome, we performed the 2-DE map from Arabidopsis cell suspensions cultured in another growth medium Murashige and Skoog (M-S) and 327 major spots were identified. Using PDQuest imaging analysis, significant increases in the amount of several housekeeping enzymes, stress/defense proteins, and heat shock proteins were found in M-S medium. Modified expression of certain proteins and detection of new isoforms involved in nitrate assimilation, nitrogen, and sulfur metabolism were also observed in the M-S medium. This study provides the first 2-DE maps of the soluble proteome of Arabidopsis cell suspensions. The comparative analysis of the Arabidopsis proteome in respect to different nutrient supplies shows that the culture medium may significantly influence the expression pattern of major soluble proteins in Arabidopsis cells. This work also constitutes an important step for further proteomic analysis concerning cell responses to abiotic or biotic stresses.     

Proteomics analysis of in vitro protein methylation during Src-induced transformation

Src, a nonreceptor tyrosine kinase, was the first oncogene identified from an oncogenic virus. Mechanistic studies of Src-induced transformations aid in understanding the pathologic processes underlying tumorigenesis and may provide new strategies for cancer therapy. Although several pathways and protein modifications are reportedly involved in Src-induced transformation, the detailed mechanisms of their regulation remain unclear. Protein methylation is an important PTM that is widely involved in cellular physiology. In this study, we determined if protein methylation was involved in Src activation and which methylated proteins were associated with this activity. Using in vitro methylation and 2-DE analysis of viral Src (v-Src)-transformed rat kidney epithelial cells (RK3E), several known and novel methylated proteins were identified based on their changes in methylation signal intensity upon transformation. Among these, elongation factor 2 (EF-2), heterogeneous nuclear ribonucleoprotein K (hnRNP K), and β-tubulin protein expressions remained unchanged, indicating that their altered methylation levels were due to Src activation. In addition, the altered expression of β-actin, vimentin, and protein phosphatase 2, catalytic subunit (PPP2C) as well as protein phosphatase 2, catalytic subunit methylation were also confirmed in RK3E cells transformed with a human oncogenic Src mutant (Src531), supporting their association with Src-induced transformation in human cancer. Together, we showed putative involvement of protein methylation in Src activation and our identification of methylated proteins provides important targets for extensively studying Src-induced transformations.     

Protein pre-fractionation in detergent-polymer aqueous two-phase systems for facilitated proteomic studies of membrane proteins

Pre-fractionation of a complex mixture of proteins increases the resolution in analytical separations of proteins from cells, tissues or organisms. Here we demonstrate a novel method for pre-fractionation of membrane proteins by a detergent-based aqueous two-phase system. Membrane proteins are strongly under-represented in proteomic studies based on two-dimensional electrophoresis (2-DE). As a model system, we have isolated mitochondria from the yeast Saccharomyces cerevisiae. Mitochondrial proteins were fractionated in an aqueous two-phase system consisting of the polymer poly(ethylene glycol) and either of two commonly used non-ionic detergents, Triton X-114 or dodecyl maltoside (DDM). Soluble proteins partitioned mainly to the polymer phase while membrane proteins were enriched in the detergent phase, as identified from one-dimensional electrophoresis (1-DE) and/or 2-DE followed by mass spectrometric analysis. Pre-fractionation was further enhanced by addition of an anionic detergent, sodium dodecyl sulfate, or a chaotropic salt, NaClO4, and by raising the pH in the system. The two-phase system pre-fractionation was furthermore combined with an alternative two-dimensional high-resolution separation method, namely ion-exchange chromatography and 1-DE. By this approach a larger number of membrane proteins could be identified compared to separation with conventional 2-DE. Thus, pre-fractionation of complex protein mixtures using the aqueous two-phase systems developed here will help to disclose larger proportions of membrane proteins in different proteomes.     

Two-dimensional electrophoretic and immunoblot analysis of cell surface proteins of spiral-shaped and coccoid forms of Helicobacter pylori

Cell surface proteins of the human gastric pathogen Helicobacter pylori extracted during different in vitro growth phases were analyzed by one- and two-dimensional gelelectrophoresis (1-DE and 2-DE) and by 2-DE immunoblot. Broth-cultured H. pylori cells were stained with an acridine-orange dye to monitor the morphological status of the organism. In 2-day-cultures, 96% of the bacterial cells were spiral-shaped and four days later a morphological switch to coccoid forms occurred. In 10-day cultures spiral-shaped forms were not found. By 1-DE, proteins with the molecular masses of 87 and 120 kDa were detected in the 2-day cultures that disappeared in cells of 12-day cultures. A protein corresponding in size to the heat shock protein (GroEl homolog, Hsp60) and a 62 kDa protein, the ureaseB-subunit, were identified in extracted proteins of 2-, 8-, and 12-day cultures. 2-DE revealed an increased number of silver-stained spots of 8-day cultures (in average 250 spots) compared with protein extracted from 2-day cells (in average 160 spots). 2-DE immunoblots performed with sera containing antibodies to major H. pylori proteins such as the A- and B-subunits of urease and the Hsp60 showed similar reactivity to surface proteins extracted from 2-, 8-, and 12-day cultures, suggesting that these proteins remain immunologically intact. Pooled sera from infected patients absorbed with spiral-shaped cells showed an almost total blocking of the antibody reactivity to extracted coccoid proteins in 2-DE immunoblot. Eighteen spots were still visible, but this reactivity probably represents a solid overexpression by the coccoid cells of Hsp60 and ureaseB proteins and is thus difficult to block.     

Assignment of human plasma polypeptides on a nondenaturing 2-D gel using MALDI-MS and PMF and comparisons with the results of intact protein mapping

Human plasma proteins were separated by 2-DE under nondenaturing conditions followed by the assignment of the CBB-stained spots using MALDI-MS and PMF, aiming to correlate the information of intact proteins with that of constituent polypeptides. A microgel system was employed to facilitate the analysis. Totally 157 spots on a nondenaturing micro-2-DE gel were numbered, the spots were excised, the proteins in the gel pieces were subjected to in-gel digestion with trypsin followed by polypeptide analysis using MALDI-MS and PMF. Two PMF algorithms, MASCOT (with Swiss-Prot database) and ProFound (with NCBInr database) were employed. A total of 153 spots out of the 157 provided significant match (p <0.05) with polypeptides in databases. Eighty spots were assigned to contain multiple (2-4) polypeptides, suggesting (i) noncovalent interaction between proteins/polypeptides, (ii) disulfide bonding of polypeptides, or (iii) overlapping of the protein locations on the gel. The results of polypeptide assignment coincided very well with the results of protein mapping previously reported, in which 33 plasma proteins were identified using blotting-immunochemical staining (Manabe, T., Takahashi, Y., Higuchi, N., Okuyama, T., Electrophoresis 1985, 6, 462-467). Further, 19 polypeptides in 25 spots were newly assigned. These results demonstrate that the techniques of MALDI-MS and PMF can be applied for analysis of proteins separated on nondenaturing 2-DE gels, providing information on their polypeptide structure. The integrated information on proteins and polypeptides would help the comprehensive understanding on the functions of complex protein systems.     

Proteome and phosphoproteome of primary cultured pig urothelial cells

Epithelial tissue lining the inner side of the urinary bladder is the most common target for bladder cancer-related diseases. Bladders of freshly slaughtered pigs were utilised for a comprehensive analysis of the proteome and phosphoproteome of bladder epithelial cells. Following protein separation by 2-D gel electrophoresis and identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) the first proteome and phosphoproteome maps of pig urinary bladder epithelial cells (PUBEC) were established. A total of 120 selected protein spots were identified. By using the La(3+) enrichment method further developed in our laboratory we identified 31 phosphoproteins with minimal contamination by non-phosphopeptides. The 2-DE map of pig urothelial cells may prove as a useful tool for studies on uroepithelial biology, and the analysed phosphoproteins expression pattern, together with the whole cell proteome, will be helpful for identifying the proteins involved in bladder-related diseases.     

温敏核不育系水稻株1S及其矮秆突变体SV14茎的蛋白质组学比较

采用双向凝胶电泳对温敏核不育水稻株1S和其矮秆突变体SV14的茎(穗颈下第1节和第2节)蛋白进行了分离, 通过银染显色, 获得了分辨率和重复性较好的双向电泳图谱. 选取了26个蛋白质点采用MALDI-TOF-MS进行肽质谱指纹图分析, 最终有12个蛋白质点得到了可靠鉴定. 其中在SV14中相对于株1S上调的仅有OSJNBa0039C07.13 蛋白, 其它蛋白均表现为下调. 这些差异蛋白按照功能可分为4类: (1) 能量代谢相关蛋白; (2) 次生代谢相关蛋白; (3) 调控蛋白; (4) 未知蛋白. 对光合系统Ⅱ氧延伸复合物蛋白质前体2, 果糖二磷酸醛缩酶, UDP-葡糖醛酸脱羧酶对应的基因进行了半定量RT-PCR分析, 发现这几个基因与蛋白质的表达不一致, 可能是RNA发生了翻译后修饰而减少了蛋白表达量的结果. 这些差异蛋白很可能与水稻矮化有关, 为水稻矮秆基因的寻找提供了另一个有效途径.     

Thermal denaturation produced degenerative proteins and interfered with MS for proteins dissolved in lysis buffer in proteomic analysis

In 1-DE, proteins were traditionally mixed with the standard Laemmli buffer and boiled for several minutes. Recently, proteins dissolved in lysis buffer were used to produce better-resolved 2-DE gels, but thermal denaturation procedure still remained in some proteomic analysis. To determine the detailed effects of thermal denaturation on SDS-PAGE and MS, both 1-DE and 2-DE were performed using proteins heated at 100°C for different periods of time, and 17 protein bands/spots were positively identified by MALDI TOF/TOF MS/MS. Protein profiles on both 1-DE and 2-DE gels changed obviously and more polydisperse bands/spots were observed with increased heating time for over-heated samples. Based on these observations, an alternative protein marker-producing method was designed by directly dissolving protein standards without BSA into lysis buffer. This new kind of protein marker could be stored at room temperature for a long time, thus was more convenient for using and shipping. The identification of 17 proteins via MS and comparison of their identities revealed MASCOT-searched scores, number of both matched peptides, total searched peptides and sequence coverage became progressively lower with increasing denaturation intensity, probably due to the interference of thermal denaturation on trypsin cleavage efficiency and produced redundant modified peptides. Therefore, it was concluded that thermal denaturation not only changed the protein profiles and produced more polydisperse protein bands/spots, but also heavily interfered with the subsequent MS analysis, hence not recommended in future proteomic analysis for proteins dissolved in lysis buffer.     

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.     

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.     

Two-dimensional electrophoresis of liver proteins: characterization of a drug-induced hepatomegaly in rats

Two-dimensional electrophoresis (2-DE) of liver proteins was applied to further characterize an unusual drug-induced increase in hepatocellular rough endoplasmic reticulum (RER) in Sprague-Dawley rats given a substituted pyrimidine derivative. Absolute liver weights of drug-treated rats (9.9 +/- 0.4 g) increased above vehicle-treated controls (7.2 +/- 0.2 g) by 37%. Light microscopy revealed diffuse granular basophilia of the hepatocellular cytoplasm, uncharacteristic of hepatocytes and suggested cells rich in ribosomes, which was confirmed by electron microscopy. Immunostaining for cell proliferation, viz., 5-bromo-2'-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA), indicated marked hepatocellular proliferative activity. 2-DE of solubilized liver using an ISO-DALT gel system indicated significant (p<0.001) quantitative changes in at least 17 liver proteins (12 increased, 5 decreased) compared to controls. The protein with the largest increase was homologous to acute-phase reactant, contrapsin-like protein inhibitor-6. Other markedly upregulated proteins were methionine adenosyltransferase, a catalyst in methionine/ATP metabolism and mitochondrial HMG-CoA synthase, involved in cholesterol synthesis. The complementary strategies of 2-DE coupled either with database spot mapping or protein isolation and amino acid sequencing successfully identified a subset of proteins from xenobiotic-damaged rodent livers, the expression of which differed from controls. However, the current bioinformatics platform for rodent hepatic proteins and limited knowledge of specific protein functionality restricted application of this proteomics profile to further define a mechanistic basis for this unusual hepatotoxicity.     

Analysis on heat stress-induced hyperphosphorylation of stathmin at serine 37 in Jurkat cells by means of two-dimensional gel electrophoresis and tandem mass spectrometry

Two-dimensional gel electrophoresis (2-DE) and tandem mass spectrometry were successfully used for determination of a phosphorylation site of stathmin induced by heat stress to Jurkat cells of a human T lymphoblastic cell line. The cells were incubated for 30 min at 41 degrees C up to 45 degrees C in a serum free 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered culture medium. The intracellular soluble proteins were separated by 2-DE, and some of the proteins increased their abundance by heat stress. Those proteins were identified to be calmodulin, protein kinase C substrate, thymosin beta-4 and F-actin capping protein beta-subunit by peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). On the contrary, protein phosphatase 2C gamma-isoform, nucleophosmin, translationally controlled tumor protein, Rho GDP-dissociation inhibitor-1, eukaryotic translation initiation factors 5A and 3A subunit 2, ubiquitin-like protein SMT 3B and chloride intracellular channel protein-1 were decreased their abundance. A protein spot of M(r) 18,000 and pI 5.9 was markedly increased at temperatures higher than 43 degrees C at which the cells were led to apoptosis. The spot was identified to be stathmin of a signal relay protein which has a function of sequestering microtubule. MALDI-quadrupole ion trap (QIT)-TOF-MS/MS and immunoblotting with a monoclonal antibody specific for a phosphorylation site of stathmin showed that the spot was a phosphorylated stathmin at serine 37 (Ser 37). The phosphorylation was suppressed by treatment of cells with olomoucine of an inhibitor specific for cyclin dependent kinase (Cdk-1). These results strongly suggest that heat stress activates Cdk-1 which phosphorylates Ser 37 on the stathmin molecule. The phosphorylation may cause the functional loss of stathmin for dynamic microtubule assembly and leads Jurkat cells to cell cycle arrest and apoptosis.     

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.     

Comprehensive proteome analysis of mouse liver by ampholyte-free liquid-phase isoelectric focusing

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.     

3-磷酸甘油醛脱氢酶与酵母线粒体基因组维持蛋白Mgm101的相互作用

采用酵母双杂交方法, 以Mgm101p为诱饵, 筛选酵母cDNA文库. 分离鉴定15个与Mgm101p相互作用的蛋白因子, 其中5个阳性克隆均为GPD1编码的3-磷酸甘油醛脱氢酶(GAPDH). 克隆了GPD1在 S. cerevisiae的同系物ScTDH2基因, 进行绿色荧光蛋白GFP标记、 亚细胞组分分离和蛋白质印迹分析, 结果表明, GAPDH除了在细胞质为糖酵解酶的主要作用外, 可能为多功能蛋白, 在酵母线粒体中与Mgm101p相互作用参与线粒体DNA维持的生物过程.     

Enhanced Production of Citric Acid in Yarrowia lipolytica by Triton X-100

Various chemical surfactants could affect permeability of yeast cells. In this study, effects of the surfactant addition upon yeast cells permeability and citric acid (CA) production by Yarrowia lipolytica strains DSM 3286 and M7 were investigated. The addition of Triton X-100 increased 1.4-1.8-fold of the maximum CA quantity achieved for both strains, with final CA concentrations ranging between 75-85 g/l that correspond to CA conversion yields per unit of glucose consumed of ~0.80-0.84 g/g. Scanning electron micrographs of yeast cells showed that the cells treated with Triton X-100 had altered cell structure and were smaller and narrower compared with the non-treated ones. The results showed that Triton X-100 could be used in order to increase the efficiency of CA production by Y. lipolytica strains.     

Identification of proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing as a prefractionation step followed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation mass spectrometry

Cerebrospinal fluid (CSF) is in close proximity to the brain and changes in the protein composition of CSF may be indicative of altered brain protein expression in neurodegenerative disorders. Analysis of brain-specific proteins in CSF is complicated by the fact that most CSF proteins are derived from the plasma and tend to obscure less abundant proteins. By adopting a prefractionation step prior to two-dimensional gel electrophoresis (2-DE), less abundant proteins are enriched and can be detected in complex proteomes such as CSF. We have developed a method in which liquid-phase isoelectric focusing (IEF) is used to prefractionate individual CSF samples; selected IEF fractions are then analysed on SYPRO-Ruby-stained 2-D gels, with final protein identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). To optimise the focusing of the protein spots on the 2-D gel, the ampholyte concentration in liquid-phase IEF was minimised and the focusing time in the first dimension was increased. When comparing 2-D gels from individual prefractionated and unfractionated CSF samples it is evident that individual protein spots are larger and contain more protein after prefractionation of CSF. Generally, more protein spots were also detected in the 2-D gels from prefractionated CSF compared with direct 2-DE separations of CSF. Several proteins, including cystatin C, IgM-kappa, hemopexin, acetyl-coenzyme A carboxylase-alpha, and alpha-1-acid glycoprotein, were identified in prefractionated CSF but not in unfractionated CSF. Low abundant forms of posttranslationally modified proteins, e.g. alpha-1-acid glycoprotein and alpha-2-HS glycoprotein, can be enriched, thus better resolved and detected on the 2-D gel. Liquid-phase IEF, as a prefractionation step prior to 2-DE, reduce sample complexity, facilitate detection of less abundant protein components, increases the protein loads and the protein amount in each gel spot for MALDI-MS analysis.     

Differential expression of phosphatidylethanol-amine-binding protein in rat hepatoma cell lines: analyses of tumor necrosis factor-alpha-resistant cKDH-8/11 and -sensitive KDH-8/YK cells by two-dimensional gel electrophoresis

To determine intracellular factors influencing the sensitivity of cancer cells to tumor necrosis factor-alpha (TNF-alpha), we studied the expression of intracellular proteins in TNF-alpha-resistant cKDH-8/11 and -sensitive KDH-8/YK rat hepatoma cell lines using the technique of two-dimensional gel electrophoresis (2-DE). From the 2-DE patterns, it was demonstrated that TNF-alpha-resistant cKDH-8/11 cells had increased levels of protein of molecular weight (Mr) 22 500 and isoelectric point (pI) 5.2, compared with TNF-alpha-sensitive KDH-8/YK cells. Therefore, we excised cyanogen bromide (CNBr) fragments of proteins in the spot for N-terminal sequencing. Microsequencing for the CNBr fragments identified the protein as rat phosphatidylethanolamine-binding protein. These findings suggest that the intracellular phosphatidylethanolamine-binding protein could be one of the factors responsible for the resistance of cKDH-8/11 cells to TNF-alpha-induced cell death.     

The dynamic range of protein expression: a challenge for proteomic research

Proteomic research, for its part, is benefiting enormously from the last decade of genomic research as we now have archived, annotated and audited sequence databases to correlate and query experimental data. While the two-dimensional electrophoresis (2-DE) gels are still a central part of proteomics, we reflect on the possibilities and realities of the current 2-DE technology with regard to displaying and analysing proteomes. Limitations of analysing whole cell/tissue lysates by 2-DE alone are discussed, and we investigate whether extremely narrow p/ranges (1 pH unit/25 cm) provide a solution to display comprehensive protein expression profiles. We are confronted with a challenging task: the dynamic range of protein expression. We believe that most of the existing technology is capable of displaying many more proteins than is currently achievable by integrating existing and new techniques to prefractionate samples prior to 2-DE display or analysis. The availability of a "proteomics toolbox", consisting of defined reagents, methods, and equipment, would assist a comprehensive analysis of defined biological systems.