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.     

Human bronchoalveolar lavage fluid protein two-dimensional database: study of interstitial lung diseases

Recently, we published an analytical two-dimensional electrophoresis (2-DE) protein map of human bronchoalveolar lavage fluid (BALF) using a pool of BALFs from various patients. In this report, the effect of lung disorders on the protein composition of the lung epithelial lining fluid was investigated by 2-DE of BALFs from individual patients with well-defined interstitial lung diseases: sarcoidosis, idiopathic pulmonary fibrosis (IPF) and hypersensitivity pneumonitis (HP), using improved experimental conditions. On these gels, about 600-1000 stained protein spots could be identified in a BALF sample containing 25 microg of protein, and our original human BALF protein database has, therefore, been considerably extended. Altogether, 429 protein spots corresponding to 66 different proteins (including isoforms, protein subunits and fragments) were identified by microsequence analysis and by matching with a human blood plasma 2-DE protein map available in the SWISS-2DPAGE database. A human 2-DE BALF database was established and is available on the World Wide Web (http://www.umh.ac.be/-biochim/proteomic.htm+ ++). The significance of the modifications observed between the different lung pathologies is discussed with the aim of understanding the mechanistic bases of lung disease pathogenesis and finding new potential lung markers of disorders.     

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.     

Towards two-dimensional electrophoresis mapping of the cerebrospinal fluid proteome from a single individual

We test the ability of state-of-the-art two-dimensional electrophoresis (2-DE) technology to enable the proteome mapping of ante mortem cerebrospinal fluid (CSF) from a single individual. Using the sensitive technologies of a fluorescent protein stain and fluorescence laser scanning of 2-DE gels, combined with matrix-assisted laser desorption/ionization-time of flight/time of flight-mass spectrometry (MALDI-TOF/TOF-MS) for protein identification, a highly detailed 2-DE map of the CSF proteome was created. The 2-DE map contains 600 identified spots representing 82 different proteins. Of the 82 proteins identified, 25 have not appeared in any previously published 2-DE map of CSF, and 11 have not been previously reported to exist in CSF. Most of the identifications originate from an ante mortem CSF sample collected from a single hydrocephalus patient. A supplemental map created from neurologically normal patients is also presented. A webpage with protein identification and scoring information from both maps is available at http://www.leelab.org/csfmap.     

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.     

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.     

Towards a two-dimensional database of common human proteins.

A protein pattern of common human proteins was constructed by comparing the two-dimensional gel electrophoresis (2-DE) protein patterns from five cell lines of different germ layers. Total cell lysate and the isolated and purified nuclei of each cell line were separated by parallel electrophoresis runs in a multiple casting system of highest reproducibility. The computerized image analysis of the digitized 2-DE gels revealed a master protein pattern for each cell line. By comparison of all master protein patterns a 2-DE protein map of only common human proteins was constructed as a basis for a new 2-DE database. In a first step we have started characterizing a number of spots by microsequencing, amino acid composition analysis, and mass spectroscopy.     

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.     

Analysis of protein/polypeptide interactions in human plasma using nondenaturing micro-2-DE followed by 3-D SDS-PAGE and MS

Previously, we have reported on the analysis of human plasma proteins on a nondenaturing micro-2-DE (mu2-DE) gel, using in-gel digestion followed by MALDI-MS and PMF [1]. Many of the spots on the mu2-DE gel showed apparent masses much larger than the calculated masses of their assigned polypeptides, suggesting noncovalent or covalent interactions between the polypeptides. In the present study, we aimed to further analyze the plasma protein spots on a nondenaturing mu2-DE gel, on which protein/polypeptide interactions have been suggested. The proteins in the spots were extracted under alkaline conditions and subjected to 3-D separation using SDS-PAGE in microslab gel format (muSDS gel) with or without the sample treatment of reduction-alkylation. The clear bands in each lane of the muSDS gels demonstrated the successful extraction of proteins from the relevant gel spot and visualized the relative contents of the polypeptides in the spot. Most of the bands were assigned by in-gel digestion followed by MALDI-MS and PMF (MASCOT/Swiss-Prot). The large discrepancy between the apparent mass value of a protein spot and the estimated mass values of the polypeptide bands on a nonreducing muSDS gel strongly suggested noncovalent polypeptide interactions. The differences in the polypeptide separation patterns on the muSDS gels, between with and without the treatment of reduction-alkylation, confirmed polypeptide disulfide bonding. The method employed here, aiming to integrate information on the proteins separated on nondenaturing 2-DE gels with that on the interactions between polypeptides, would help the comprehensive understanding of complex protein systems.     

A protein extraction method compatible with proteomic analysis for the euhalophyte Salicornia europaea

Protein extraction from plants like the halophyte Salicornia europaea has been problematic using standard protocols due to high concentrations of salt ions in their cells. We have developed an improved method for protein extraction from S. europaea, which allowed us to remove interfering compounds and salt ions by including the chemicals borax, polyvinylpolypyrrolidone, and phenol. The comparative study of this method with several other protocols using NaCl-treated S. europaea shoots demonstrated that this method gave the best distinction of proteins on 2-DE gels. This protocol had a wide range of applications as high yields and good distinction of 1-DE gels for proteins isolated from twelve other plants were rendered. In addition, we reported results of 2-DE using the recalcitrant tissue of the S. europaea roots. We also demonstrated that this protocol is compatible with proteomic analysis as eight specific proteins generated by this method have been identified by MS. In conclusion, our newly developed protein extraction protocol is expected to have excellent applications in proteomic studies of halophytes.     

A two dimensional electrophoresis database of a human Jurkat T-cell line

About 2000 protein spots of human Jurkat T-cells were detected by high resolution two-dimensional gel electrophoresis (2-DE) and were characterized in terms of their isoelectric point and molecular mass. A 2-DE database was constructed and is available at http://www.mpiib-berlin.mpg.de/2D-PAGE/. At present the database contains 67 identified protein spots. These proteins were identified after tryptic digestion by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-mass spectrometry (DE-MALDI-MS). Proteins with a sequence coverage of at least 30% were introduced in the database. This sequence coverage could not always be obtained by using only the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) for the mass analysis. Therefore, an additional mass spectrum was recorded by using 2,5-dihydroxybenzoic acid (DHB). Usually, additional mass peaks were detected and together with the mass spectrum of CHCA this resulted in the desired sequence coverage.     

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.     

Human proteome enhancement: high-recovery method and improved two-dimensional map of colostral fat globule membrane proteins.

The human milk fat globule membrane protein composition is still largely unknown, although it counts for 2-4% of the total milk protein content and contains several important biologically active components. The aim of this work was to create a two-dimensional electrophoresis (2-DE) map of the human milk fat globule membrane proteins, both integral and membrane-associated, and to identify and characterize as many protein components as possible. A new protocol for the solubilization and extraction of the human milk fat globule membrane proteins with a double extraction procedure is presented, and the results compared with the extraction methods reported in the literature. The proteins were separated, in the first dimension, by isoelectric focusing (IEF) in the pH range 3-10 on strips of 13 cm length and, in the second dimension, by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 11.5% T homogeneous gels. A reproducible 2-DE map of integral and membrane-associated proteins was obtained and the first 23 spots, representing the major components, were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis and/or by amino acid sequencing.     

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).     

Alteration of protein composition in mouse thymocytes by signals through T-cell receptor

To avoid destructive autoimmunity, T-cell precursors (thymocytes) expressing autoreactive T-cell receptor are deleted in the thymus via an apoptotic process by the signals from the T-cell receptor-CD3 complexes. In order to analyze the apoptotic mechanism, we established a cell-free system using the lysates from mouse thymocytes treated in vivo with anti-CD3 monoclonal antibody (mAb). The soluble cytosolic high molecular mass protein fraction from the anti-CD3-treated thymocytes revealed an activity that directly induces nuclear apoptotic morphological changes and DNA fragmentation. This fragmentation activity was not observed in the fraction from the thymocytes without anti-CD3 treatment. Proteins in both fractions were separated by two-dimensional electrophoresis. The silver-stained gels revealed differences in protein spots. These protein spots were identified by database searching of mass spectrometric (MS) and tandem mass spectrometric (MS/MS) data obtained from in-gel tryptic digests of the spots, using an integrated system of liquid chromatography/electrospray ionization/ion-trap mass spectrometry. In this study, the high mobility group protein HMG2 was identified as one of the cytosolic proteins that is increased by the signals from the T-cell receptor, and heterogeneous nuclear ribonucleoprotein A2/B1 and glyceraldehyde 3-phosphate dehydrogenase were found to be decreased by the signals.     

Two-dimensional electrophoresis of the fatty acid binding protein from human heart: evidence for a thiol group which can form an intermolecular disulfide bond

A 100,000 g supernatant from human heart muscle, containing cytosolic proteins with some contaminating plasma proteins, was analyzed for fatty acid binding protein (FABP) by two-dimensional electrophoresis (2-DE) using isoelectric focusing under nondenaturing conditions in the first dimension. FABP purified from human heart muscle was found to comigrate with a major spot in 2-DE gels of the supernatant. This spot was comparable with those of the myoglobins in staining intensity. When purified FABP was charged with [3H]palmitate and subjected to nondenaturing 2-DE, radioactivity always comigrated with this protein. Under denaturing and reducing conditions in the second dimension, FABP was found to have a pI of 5.3 and an apparent molecular weight of 15,000. Isoforms of FABP, reported here for the first time to occur in human heart muscle, were observed as minor spots focusing at pH 5.1 and 5.7. When electrophoresis in the second dimension was carried out under denaturing but nonreducing conditions, an additional protein appeared at pH 5.3 with an apparent molecular weight of about 30,000. This protein was identified as a dimer of FABP and evidence for the involvement of an intermolecular disulfide bond in this dimerization is presented.     

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.     

The establishment of a human liver nuclei two-dimensional electrophoresis reference map

This short communication describes the establishment of a two-dimensional electrophoresis (2-DE) reference map of nuclear proteins isolated from human liver. The human liver nuclei 2-DE reference map contains 1497 spots. In an initial identification study using peptide mass fingerprinting as a means of protein identification we were able to identify 26 spots corresponding to 15 different proteins. The human liver nuclei 2-DE reference map is now included in the SWISS-2DPAGE database, which can be accessed through the ExPASy server (http://www.expasy.ch/ch2d/).     

Detection of immunogenic parasite and host-specific proteins in the sera of active and chronic individuals infected with Toxoplasma gondii

Toxoplasma gondii infection in pregnant women may result in abortion and foetal abnormalities, and may be life-threatening in immunocompromised hosts. To identify the potential infection markers of this disease, 2-DE and Western blot methods were employed to study the parasite circulating antigens and host-specific proteins in the sera of T. gondii-infected individuals. The comparisons were made between serum protein profiles of infected (n=31) and normal (n=10) subjects. Antigenic proteins were identified by immunoblotting using pooled sera and monoclonal anti-human IgM-HRP. Selected protein spots were characterised using mass spectrometry. Prominent differences were observed when serum samples of T. gondii-infected individuals and normal controls were compared. A significant up-regulation of host-specific proteins, α(2)-HS glycoprotein and α(1)-B glycoprotein, was also observed in the silver-stained gels of both active and chronic infections. However, only α(2)-HS glycoprotein and α(1)-B glycoprotein in the active infection showed immunoreactivity in Western blots. In addition, three spots of T. gondii proteins were detected, namely (i) hypothetical protein chrXII: 3984434-3 TGME 49, (ii) dual specificity protein phosphatase, catalytic domain TGME 49 and (iii) NADPH-cytochrome p450 reductase TGME 49. Thus, 2-DE approach followed by Western blotting has enabled the identification of five potential infection markers for the diagnosis of toxoplasmosis: three are parasite-specific proteins and two are host-specific proteins.     

A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis

Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Three phenol-free protein extraction procedures for 2-DE were compared and evaluated on apple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone precipitation was found to be the most effective protocol. The results from SDS-PAGE and 2-DE analysis showed high quality proteins. More than 500 apple polypeptides were separated on a small scale 2-DE gel. The successful protocol was further tested on banana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues, respectively. To demonstrate the quality of the extracted proteins, several protein spots from apple and banana peels were cut from 2-DE gels, analyzed by MS and have been tentatively identified. The protocol described in this study is a simple procedure which could be routinely used in proteomic studies of many types of recalcitrant fruit tissues.