Prediction of translocation and cleavage of heterogeneous ribonuclear proteins and Rho guanine nucleotide dissociation inhibitor 2 during apoptosis by subcellular proteome analysis

Jurkat T cells induced to undergo apoptosis by the CD95(Fas/Apo-1) pathway were investigated by proteome analysis. The most prominent differing protein spots of apoptotic and nonapoptotic cells were identified as various heterogeneous ribonuclear proteins (hnRNPs) and Rho guanin nucleotide dissociation inhibitor (GDI) 2. In apoptotic cells, four spots slightly differing in molecular mass and/or isoelectric point were identified as Rho GDI 2 with the mass and pI as expected after caspase-3 cleavage near the N-terminus. Subcellular proteome analysis revealed that Rho GDI 2 was highly enriched in the cytosolic fraction, present in minor amounts in the nuclear fraction and absent from the mitochondrial fraction. In apoptotic cells however, the spots representing processed and modified Rho GDI 2 were found in the cytosol, in the nucleus and also the mitochondria at different spot positions. In addition, twelve different hnRNPs were identified to be altered after induction of cell death of which hnRNPs A/B, D, F, H, I and L were hitherto unknown to be modified during apoptosis. Most of the hnRNP spots were found in the nucleus of nonapoptotic cells, whereas these proteins, either modified or unmodified, relocated to the cytosol and/or the mitochondria in apoptotic cells. Our results demonstrate that modification of proteins during apoptosis is often accompanied by their relocalisation between cellular compartments.     

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

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

Predominant identification of RNA-binding proteins in Fas-induced apoptosis by proteome analysis

Proteome analysis of Jurkat T cells was performed in order to identify proteins that are modified during apoptosis. Subtractive analysis of two-dimensional gel patterns of apoptotic and nonapoptotic cells revealed differences in 45 protein spots. 37 protein spots of 21 different proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. The hnRNPs A0, A2/B1, A3, K, and R; the splicing factors p54(nrb), SRp30c, ASF-2, and KH-type splicing regulatory protein (FUSE-binding protein 2); and alpha NAC, NS1-associated protein 1, and poly(A)-binding protein 4 were hitherto unknown to be involved in apoptosis. The putative cleavage sites of the majority of the proteins could be calculated by the molecular masses and isoelectric points in the two-dimensional electrophoresis gel, the peptide mass fingerprints, and after translation by treatment with recombinant caspase-3. Remarkably, 15 of the 21 identified proteins contained the RNP or KH motif, the best characterized RNA-binding motifs.     

Proteomic analysis of rat pheochromocytoma PC12 cells

PC12 cell line is well documented and widely applied as many kinds of models in neurobiological and neurochemical studies. Yet a thorough proteomic analysis has not been performed so far. Here we report the construction of a large-scale 2-D protein database for PC12 cells. The proteins extracted from PC12 cells were separated by 2-DE and identified by MALDI-TOF/TOF MS. A total of 1080 protein spots, excised from three different 2-D gels, were identified with high confidence. These proteins represent 474 different gene products, mainly binding proteins and enzymes. Three hundred and seven identified protein spots were located in the low-molecular weight region below 20 kDa. This database today represents one of the largest 2-D databases for higher eukaryotic cell proteomes and for low-molecular weight proteins. In addition, fragment ion spectra obtained by TOF/TOF confirmed that calcylin in PC12 cells was N-acetylated. The database of PC12 proteome is expected to be a powerful tool for neuroscientists.     

Shotgun proteome analysis of protein cleavage in apoptotic cells

A new shotgun proteomics approach was employed to identify degraded proteins. Jurkat T-cells were induced to undergo apoptosis by Fas (CD95/Apo-1) stimulation. The proteins were separated by large (30 cm) sodium dodecyl sulphate-polyacrylamide gel electrophoresis and identified by liquid chromatography-tandem mass spectrometry after digestion of 100 gel slices with trypsin. The molecular masses of the individual gel slices were calculated through the known theoretical masses of the identified proteins. Proteins were defined as degradation candidates if either the empirical determined molecular mass was at most 80% of the theoretical value, or if proteins were identified in clearly different gel slices. In this manner, the degradation of 11 already identified apoptosis-modified proteins was confirmed and nine until now unknown degradation candidate proteins identified. Degradation during apoptosis must be verified by additional techniques such as in vitro caspase assays as shown for nucleolin and Rho GDI 2. The results presented confirm the suitability of a shotgun approach for the identification of putative protease targets.     

Separation of human erythrocyte membrane associated proteins with one-dimensional and two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry

A classical proteomic analysis was used to establish a reference map of proteins associated with healthy human erythrocyte ghosts. Following osmotic lysis and differential centrifugation, ghost proteins were separated by either one-dimensional gel electrophoresis (1-DE) or two-dimensional gel electrophoresis (2-DE). Selected protein bands or spots were excised and trypsinized before mass spectrometric analyses and data mining was performed using the SWISS-PROT and NCBI nonredundant databases. A total of 102 protein spots from a 2-D gel were successfully identified. These corresponded to 59 distinct polypeptides with the remaining 43 being isoforms. As for the 1-D gel, 44 polypeptides were identified, of which 19 were also found on the 2-D gel. Most of the 19 common polypeptides were membrane cytoskeletal proteins that are often referred to as the "band" proteins. The remaining 25 polypeptides that were found exclusively on 1-D gels were proteins with high hydrophobicity (e.g., sorbitol dehydrogenase and glucose transporter) and high molecular mass (e.g., Kell blood group glycoprotein and Janus-kinase 2). A higher number of signaling proteins was also identified on 1-D gels compared to 2-D gels. These included Ras, cAMP dependent protein kinase and TGF-beta receptor type 1 precursor.     

Immunoproteomics of extracellular proteins of Chinese virulent strains of Streptococcus suis type 2

Streptococcus suis type 2 (SS2) is a porcine zoonotic pathogen with worldwide distribution, and lacking suitable vaccine and virulent maker were bottleneck to control this infection. An immunoproteomic assay was used to identify antigenic proteins from the total extracellular proteins of the virulent Chinese SS2 strain ZY05719. The convalescent serum of a specific pathogen free (SPF) mini-pig recognized nine protein spots on PVDF membrane. Antigenic proteins on a duplicate gel, as well as those with a similar placement of extracellular proteins from another virulent strain (HA9801) and an avirulent strain (T15) on 2-D gels, were excised and identified by MALDI-TOF-MS. PMF of the protein spots were performed using the MASCOT server. Two proteins were found in all three strains. Comparative proteomic analysis between the two virulent strains and the avirulent strain revealed nine differential proteins, eight of which were successfully identified. Genes for six of the differentially expressed proteins were found in both virulent strains, and of those were present in the avirulent stain.     

Toward a comprehensive quantitative proteome database: protein expression map of lymphoid neoplasms by 2-D DIGE and MS

Using 2-D DIGE, we constructed a quantitative 2-D database including 309 proteins corresponding to 389 protein spots across 42 lymphoid neoplasm cell lines. The proteins separated by 2-D PAGE were identified by MS and assigned to the expression data obtained by 2-D DIGE. The cell lines were categorized into four groups: those from Hodgkin's lymphoma (HL) (4 cell lines), B cell malignancies (19 cell lines), T cell malignancies (16 cell lines), and natural killer (NK) cell malignancies (3 cell lines). We characterized the proteins in the database by classifying them according to their expression level. We found 28 proteins with more than a 2-fold difference between the cell line groups. We also noted the proteins that allowed multidimensional separation to be achieved (1) between HL cells and other cells, (2) between the cells derived from B cells, T cells and NK cells, and (3) between HL cells and anaplastic large cell lymphoma cells. Decision tree classification identified five proteins that could be used to classify the 42 cell lines according to differentiation. These results suggest that the quantitative 2-D database using 2-D DIGE will be a useful resource for studying the mechanisms underlying the differentiation phenotypes of lymphoid neoplasms.     

A proteome reference map for Vibrio cholerae El Tor

A proteome reference map has been constructed for Vibrio cholerae El Tor, in the pI range of 4.0 to 7.0. The map is based on two-dimensional gels (2-D) and the identification, by peptide mass fingerprint, of proteins in 94 spots, corresponding to 80 abundant proteins. Two strains are compared, strain N16961 and a Latin American El Tor strain C3294. The consensus map contains 340 spots consistently seen with both strains grown in Luria-Bertani broth (LB) or minimal M9 medium. The results were obtained from nine gels run with 18 cm immobilized pH gradient strips and precast gels. The 2-D gels were anchored to real N16961 proteins identified by mass spectrometry. Various energy metabolism components and periplasmic ATP-binding cassette (ABC) transporter proteins were identified among the abundant proteins. Two isoforms of OmpU were found. Five operons are proposed and seven hypothetical proteins were experimentally confirmed. Comparisons are made with protein 2-D gels for a classical strain and to microarray analysis available for the N16961 El Tor strain. New results were obtained from the proteome analysis, indicating an abundance of periplasmic ABC transporter proteins not found in microarray studies.     

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.     

The proteins secreted by Trichomonas vaginalis and vaginal epithelial cell response to secreted and episomally expressed AP65

We showed recently that contact of human vaginal epithelial cells (VECs) by Trichomonas vaginalis and incubation with trichomonad proteins in conditioned medium induced expression of VEC genes. We performed 2-D SDS-PAGE followed by MALDI-TOF to identify the major secreted proteins. Based on protein abundance and separation of spots in 2-D gels, 32 major secreted proteins were examined, which gave 19 proteins with accession numbers. These proteins included known secreted cysteine proteinases. In addition, other secreted proteins were enzymes of carbohydrate metabolism, adhesin protein AP65, heat shock proteins, thioredoxin reductase and coronins. We confirmed that the secreted trichomonad proteins induced expression of VEC genes, including interleukin 8 (IL-8), COX-2 and fibronectin. Purified AP65 added to VECs had a pronounced effect only on IL-8 gene expression, which was inhibited in the presence of 12G4 monoclonal antibody to AP65. Moreover, AP65 expressed episomally within epithelial cells was found to enhance the expression of IL-8 and COX-2. This may be the first report of analysis of the secreted proteins of T. vaginalis and of the host epithelial cell response to these proteins and to the prominent adhesin AP65.     

Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H and MHCC97-L, with different metastasis potentials

To better understand the mechanism underlying hepatocellular carcinoma (HCC) metastasis and to search for potential markers for HCC prognosis, differential proteome analysis on two HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional (2-D) gel electrophoresis followed by matrix-assisted laser desorption/time of flight mass spectrometry and liquid chromatography ion trap mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 56 protein spots showed significant differential expression in MHCC97-H and MHCC97-L cells (Student's t-test, P < 0.05) and 4 protein spots were only detected in MHCC97-H cells. Fourteen protein spots were further identified using in-gel tryptic digestion, peptide mass fingerprinting and tandem mass spectrometry. The expressions of pyruvate kinase M2, ubiquitin carboxy-terminal hydrolase L1, laminin receptor 67 kDa, S100 calcium-binding protein A4, thioredoxin and cytokeratin 19 were elevated in MHCC97-H cells. However, manganese superoxide dismutase, calreticulin precursor, cathepsin D, lactate dehydrogenase B, non-metastatic cell protein 1, cofilin 1 and calumenin precursor were down-regulated in MHCC97-H cells. Intriguingly, most of these identified proteins have been reported to be associated with tumor metastasis. The functional implications of alterations in the levels of these proteins are discussed.     

Identification of the major outer membrane proteins of Aeromonas salmonicida

Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the etiological agent of furunculosis, a serious infectious disease of salmonids. Aeromonas spp. are ubiquitous waterborne bacteria responsible for a wide spectrum of diseases among aquatic organisms and humans. Bacterial outer membrane proteins (OMPs) play a significant role in virulence as they comprise the outermost surface in contact with host cells and immune defense factors. To identify the major OMPs of A. salmonicida a proteomic analysis was undertaken using a carbonate OMP-enrichment protocol. The enriched OMP-extracts were separated by 2-dimensional electrophoresis (2-DE) and the spots identified using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) via an electrospray ionization source. In total, 76 unique proteins were identified from the 125 spots observed on the 2-D gel. The surface layer (S-layer) VapA protein dominated the A. salmonicida OMP 2-D profile, accounting for 60% of the protein on the 2-D gels. Among the other outer membrane proteins identified were at least 10 porins and various receptors involved in nutrient acquisition. Also identified in the carbonate insoluble fraction were phosphoglycerate kinase, enolase and others that lacked classical export sorting signals. The putative association of these proteins with the cell surface might provide new insights concerning the biological and pathogenic roles of these molecules in A. salmonicida infection. This work represents the first systematic attempt to characterize the cell surface of A. salmonicida.     

Nonchromatin nuclear proteins of mammalian lens epithelial cells

The nuclear matrix (NM) proteins of six tissue cultured lens epithelial cell lines and one embryonic rabbit epidermal cell line were analyzed to determine possible tissue and species specificity of these proteins. The NM proteins were isolated by the modified Penman technique. The tissue cultured cells were pulsed with [³⁵S] methionine and nuclear matrix proteins were fractionated by two-dimensional (2-D) gel electrophoresis. The 2-D gels were dried and autoradiographed. The relative abundance of spot patterns of nuclear matrix proteins of different cells were compared. The data from these experiments revealed that all the examined cell lines have distinct spot patterns, however, all of NM profile showed a spot pattern in the 45 kDa region with acidic pH. Some of these spots cross-reacted with anti-vimentin antibodies, whereas a prominent protein spot in this region did not cross react with either vimentin or actin antibodies. The observed variations in the NM protein patterns of lens epithelial cells may reflect tissue and species specificity and also a role in the regulatory properties of these nuclear proteins in the eye tissue development. J. Cell. Biochem. 64:644–650. © 1997 Wiley-Liss, Inc.     

A Quantitative Proteomic Analysis Uncovers the Relevance of CUL3 in Bladder Cancer Aggressiveness

To identify aggressiveness-associated molecular mechanisms and biomarker candidates in bladder cancer, we performed a SILAC (Stable Isotope Labelling by Amino acids in Cell culture) proteomic analysis comparing an invasive T24 and an aggressive metastatic derived T24T bladder cancer cell line. A total of 289 proteins were identified differentially expressed between these cells with high confidence. Complementary and validation analyses included comparison of protein SILAC data with mRNA expression ratios obtained from oligonucleotide microarrays, and immunoblotting. Cul3, an overexpressed protein in T24T, involved in the ubiquitination and subsequent proteasomal degradation of target proteins, was selected for further investigation. Functional analyses revealed that Cul3 silencing diminished proliferative, migration and invasive rates of T24T cells, and restored the expression of cytoskeleton proteins identified to be underexpressed in T24T cells by SILAC, such as ezrin, moesin, filamin or caveolin. Cul3 immunohistochemical protein patterns performed on bladder tumours spotted onto tissue microarrays (n = 284), were associated with tumor staging, lymph node metastasis and disease-specific survival. Thus, the SILAC approach identified that Cul3 modulated the aggressive phenotype of T24T cells by modifying the expression of cytoskeleton proteins involved in bladder cancer aggressiveness; and played a biomarker role for bladder cancer progression, nodal metastasis and clinical outcome assessment.     

达坂喜盐芽孢杆菌D-8^T在低渗冲击下的双向凝胶电泳分析

为了解革兰氏阳性中度嗜盐菌适应低渗冲击的机制,采用双向凝胶电泳技术,研究达坂喜盐芽孢杆菌(Halobacillus dabanensis)D-8T在低渗冲击下的差异蛋白表达谱。利用ImagemasterTM2D Platinum软件分析到大约650个蛋白点,大多数蛋白分子量分布在17.5~66kDa,等电点为4.0~5.9,偏酸性。在20%盐浓度中生长的D-8菌株受到0%盐浓度的低渗冲击5min及50min后34个蛋白点的表达发生改变,包括20个表达上调的点和14个点表达下调。用MALDI-TOF/MS及MASCOT软件鉴定了4个与低渗胁迫有关的蛋白,分别为热激蛋白DanK、柱状决定蛋白、青霉素结合蛋白和5-莽草酸烯醇式丙酮酸-3-磷酸合成酶。其中,热激蛋白适应低渗胁迫时表达上调为首次报道。