Differential expression of proteins in the midgut of Anopheles albimanus infected with Plasmodium berghei

The main vector for transmission of malaria in Mexico is the Anopheles albimanus mosquito. The midgut of disease-transmitting mosquitoes carries out a variety of functions that are related to blood feeding. We analyzed the midgut of A. albimanus infected with Plasmodium berghei (resistant mosquito) using a proteomic approach to identify putative short peptides that are enriched in the midgut after blood feeding. Mosquito midguts were analyzed by two-dimensional electrophoresis to determine the changes in protein profiles. We identified 21 spot proteins that are differentially expressed in the blood of mosquitoes during the immune challenge. Molecular weight of the spots varied from 13 to 36 kDa, with a broad isoelectric point range of 3.92–8.90. We identified the differentially expressed proteins using mass spectrometry and constructed a proteomic data base of the A. albimanus midgut with diverse functions, some of them proteins with digestive and immunologic functions. Identification of these proteins may have important implications for understanding the blood meal digestion process, as well as developing novel vector control strategies and understanding parasite vector interactions.     

Analysis of proteomic changes in roots of soybean seedlings during recovery after flooding

A proteomic approach was used to identify proteins involved in post-flooding recovery in soybean roots. Two-day-old soybean seedlings were flooded with water for up to 3 days. After the flooding treatment, seedlings were grown until 7 days after sowing and root proteins were then extracted and separated using two-dimensional polyacrylamide gel electrophoresis (2-DE). Comparative analysis of 2-D gels of control and 3 day flooding-experienced soybean root samples revealed 70 differentially expressed protein spots, from which 80 proteins were identified. Many of the differentially expressed proteins are involved in protein destination/storage and metabolic processes. Clustering analysis based on the expression profiles of the 70 differentially expressed protein spots revealed that 3 days of flooding causes significant changes in protein expression, even during post-flooding recovery. Three days of flooding resulted in downregulation of ion transport-related proteins and upregulation of proteins involved in cytoskeletal reorganization, cell expansion, and programmed cell death. Furthermore, 7 proteins involved in cell wall modification and S-adenosylmethionine synthesis were identified in roots from seedlings recovering from 1 day of flooding. These results suggest that alteration of cell structure through changes in cell wall metabolism and cytoskeletal organization may be involved in post-flooding recovery processes in soybean seedlings.     

Comparative immunoproteomics of identification and characterization of virulence factors from Helicobacter pylori related to gastric cancer

Helicobacter pylori is an important risk factor of gastric cancer (GC). Although many H. pylori virulence factors have been reported, the pathogenic mechanism by which H. pylori infection causes GC remains unclear. The aims of this study were to identify GC-related antigens from H. pylori and characterize their roles in the development of GC. As GC and duodenal ulcer (DU) are considered clinically divergent, we compared two-dimensional immunoblots of an acid-glycine extract of H. pylori probed with serum samples from 15 patients with GC and 15 with DU to find GC-related antigens, which were subsequently identified by mass spectrometry. Many protein spots were recognized by more than one serum, and 24 of these were better recognized by GC sera. The proteins showing higher frequency of recognition in GC group are threonine synthase, rod shape-determining protein, S-adenosylmethionine synthetase, peptide chain release factor 1, DNA-directed RNA polymerase alpha subunit, co-chaperonin GroES (monomeric and dimeric forms), response regulator OmpR, and membrane fusion protein. Of these proteins, GroES was identified as a dominant GC-related antigen with a much higher seropositivity of GC samples (64.2%, n = 95) compared with 30.9% for gastritis (n = 94) and 35.5% for DU (n = 124). GroES seropositivity was more commonly associated with antral GC than with non-antral GC (odds ratio = 2.7; 95% confidence interval, 1.1-6.7). In peripheral blood mononuclear cells, GroES stimulated production of interleukin (IL)-8, IL-6, granulocyte macrophage colony-stimulating factor, IL-1beta, tumor necrosis factor-alpha, cyclooxygenase-2, and prostaglandin E(2). Moreover when incubated with gastric epithelial cells, GroES induced expression of IL-8, cell proliferation, and up-regulation of c-jun, c-fos, and cyclin D1 but caused down-regulation of p27(Kip1). We conclude that GroES of H. pylori is a novel GC-associated virulence factor and may contribute to gastric carcinogenesis via induction of inflammation and promotion of cell proliferation.     

Comparative proteome analysis of fractions enriched for membrane-associated proteins from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica strains

The facultative intracellular pathogen Francisella tularensis is the causative agent of the serious infectious disease tularemia. Despite intensive research, the virulence factors and pathogenetic mechanisms remain largely unknown. To identify novel putative virulence factors, we carried out a comparative proteome analysis of fractions enriched for membrane-associated proteins isolated from the highly virulent subspecies tularensis strain SCHU S4 and three representatives of subspecies holarctica of different virulence including the live vaccine strain. We identified six proteins uniquely expressed and four proteins expressed at significantly higher levels by SCHU S4 compared to the ssp. holarctica strains. Four other protein spots represented mass and charge variants and seven spots were charge variants of proteins occurring in the ssp. holarctica strains. The genes encoding proteins of particular interest were examined by sequencing in order to confirm and explain the findings of the proteome analysis. Our studies suggest that the subspecies tularensis-specific proteins represent novel potential virulence factors.     

胃癌及癌旁组织定量比较蛋白质组学研究

为寻找胃癌特异的肿瘤标记物,用于胃癌临床诊断及药物治疗靶点的选择,本研究采用荧光差异显示凝胶电泳（DIGE）技术分离并筛选 Cy3、Cy5 及 Cy2 荧光素标记的胃癌及对应癌旁组织差异表达蛋白质,用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）或串联质谱技术进行鉴定并分析。结果共筛选出 33 个差异表达蛋白质点,其中 9 个蛋白质点在胃癌组织中上调,24 个蛋白质点下调。对 22 个蛋白质点采用质谱技术成功鉴定,突变结蛋白、锰超氧化物歧化酶、热休克蛋白 60等在胃癌中高表达,热休克蛋白 27、前列腺素 F 合酶、硒结合蛋白 1、锌指蛋白 160、微管蛋白 α6、真核生物翻译延伸因子 1 α1 等在胃癌组织中低表达,并筛选出 5 个未知蛋白。这些差异表达蛋白可望成为胃癌诊断的特异标记物,并与胃癌的发生、发展及预后等有关,为胃癌的诊断、发生机制的研究提供了新的思路。     

Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dye

To identify the proteomic alterations associated with carcinogenesis of hepatocellular carcinoma (HCC), we compared the protein expression profiles of nine HCC cell lines with those of primary cultured hepatocytes established from five individuals. A differential proteomic study was performed by two-dimensional difference gel electrophoresis, in which protein samples are labeled with different fluorescent dyes and separated according to their isoelectric point and molecular weight. To label the protein samples, we used a newly developed and highly sensitive fluorescent dye, which reacts with all reduced cysteine residues of proteins. Principal component analysis based on the intensity of 1238 protein spots indicated that the HCC cells and the normal hepatocytes had distinct proteomic profiles. The Wilcoxon test was used to determine the protein spots whose intensity was differentially regulated in the HCC cells compared with the normal hepatocytes, and mass spectrometric analysis was used to identify the proteins corresponding to the spots. The proteins identified are involved in cell cycle regulation, binding to a tumor-suppressor gene product, fatty acid binding, and regulation of translation. Western blotting with specific antibodies revealed the overexpression of PCNA, EB1 and E-FABP in HCC tissues compared with noncancerous tissues. Aberrant regulation of EB1 and E-FABP has not previously been implicated in the development of HCC.     

The effect of selenium enrichment on baker's yeast proteome

The use of regular yeast (RY) and selenium-enriched yeast (SEY) as dietary supplement is of interest because the Nutritional Prevention of Cancer (NPC) trial revealed that SEY but not RY decreased the incidence of prostate cancer (PC). Using two-dimensional difference in gel electrophoresis (2D-DIGE)-tandem mass spectrometry (MS/MS) approach, we performed proteomic analysis of RY and SEY to identify proteins that are differentially expressed as a result of selenium enrichment. 2D-DIGE revealed 96 candidate protein spots that were differentially expressed (p ≤ 0.05) between SEY and RY. The 96 spots were selected, sequenced by LC/MS/MS and 37 proteins were unequivocally identified. The 37 identified proteins were verified with ProteinProphet software and mapped to existing Gene Ontology categories. Furthermore, the expression profile of 5 of the proteins with validated or putative roles in the carcinogenesis process, and for which antibodies against human forms of the proteins are available commercially was verified by western analysis. This study provides evidence for the first time that SEY contains higher levels of Pyruvate Kinase, HSP70, and Elongation factor 2 and lower levels of Eukaryotic Translation Initiation Factor 5A-2 and Triosephosphate Isomerase than those found in RY.     

Comparative proteomic analysis of hearts of adult SCNT Bama miniature pigs (Sus scrofa)

This study aims to determine the effects of SCNT on cardiac development of SCNT pigs through proteomic methods. Heart proteins from three adult SCNTs and two normal reproductive Bama miniature pigs were extracted, separated, and identified via comparative proteomic methods, including two-dimensional gel electrophoresis, mass spectrometry, and Western blot. Eleven differentially expressed spots were identified as differentially expressed proteins, of which five spots were upregulated proteins such as cardiac myosin heavy chain, cathepsin D, and heat shock protein beta-1 (HSP27). By contrast, six spots were downregulated proteins such as alpha skeletal muscle and actin. The results also demonstrated that nuclear transfer might result in abnormal expression of some important proteins in hearts from SCNT pigs, and affect the cardiac development in SCNT pigs' survival.     

Proteomic signature of human cancer cells

We assessed proteomic profiles as biomarkers for monitoring cell phenotypes. Protein expression profiles were obtained by fluorescence two-dimensional difference gel electrophoresis (2-D-DIGE), in which quantitative ability is improved by labeling proteins with fluorescent dyes prior to electrophoresis. Integrated protein spot intensities were analyzed by a statistical approach. The proteomic data of two groups of cell lines: (1) adenocarcinoma (AC) cell lines derived from lung, pancreas and colon tissues and (2) lung cancer cell lines with different histological backgrounds, including AC, squamous cell carcinoma and small cell carcinoma, were assessed on the basis of prior biological information. Hierarchical clustering analysis and principal component analysis were used to divide the cell lines into subgroups on the basis of similarities between their protein expression profiles. The majority of cell lines were grouped according to their organ of origin or histological background. A machine-learning algorithm selected 32 protein spots that were responsible for the classification. The results indicate that proteomic data generated by 2-D-DIGE can provide a signature of essential cell phenotypes, suggesting that it might be possible to apply this technique to developing tumor markers that could identify the organ of origin of metastatic tumors and contribute to the differential diagnosis of lung cancer.     

Proteome analysis of the responses of Panax ginseng C. A. Meyer leaves to high light: use of electrospray ionization quadrupole-time of flight mass spectrometry and expressed sequence tag data

We performed comparative proteomic analyses in order to understand the physiological responses of ginseng (Panax ginseng C. A. Meyer) to high light (HL). As a first step, we analyzed the proteins expressed in ginseng leaves. Proteins extracted from leaves were separated by two-dimensional polyacrylamide gel electrophoresis. Protein spots were identified by tandem mass spectra analysis using electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS). We used a ginseng expressed sequence tag (EST) database as well as a nonredundant protein database from NCBI to identify proteins. Eighty-one proteins were identified using the nr protein database, 51 of which were also verified from the ginseng EST database. An additional 66 proteins were identified only from the ginseng EST database. Proteins that function in energy metabolism, protein stabilization, and protection against oxidative stress were abundant. To understand the light responses of ginseng leaves, we studied time dependent changes in expressed proteins produced by 0-4 h of HL exposure. Six HL-responsive proteins were identified: three proteins were up-regulated (cytosolic small heat-shock protein, cytosolic ascorbate peroxidase, and putative major latex-like protein) and three proteins were down-regulated (Rieske Fe/S protein, putative 3-beta hydroxysteroid dehydrogenase/isomerase-like protein, and oxygen-evolving enhancer-like protein). Our results show that the ginseng EST database combined with ESI Q-TOF MS analysis can be used to identify ginseng proteins and to elucidate the protective mechanism of ginseng against HL induced damage.     

Proteomic analysis of gingival crevicular fluid for discovery of novel periodontal disease markers

The protein composition of gingival crevicular fluid (GCF) may reflect the pathophysiology of periodontal diseases. A standard GCF proteomic pattern of healthy individuals would serve as a reference to identify biomarkers of periodontal diseases by proteome analyses. However, protein profiles of GCF obtained from apparently healthy individuals have not been well explored. As a step toward detection of proteomic biomarkers for periodontal diseases, we applied both gel-based and gel-free methods to analyze GCF obtained from healthy subjects as compared with supragingival saliva. To ensure optimized protein extraction from GCF, a novel protocol was developed. The proteins in GCF were extracted with high yield by urea buffer combined with ultrafiltration and the intensity of spots with supragingival saliva and GCF was compared using agarose two-dimensional electrophoresis. Eight protein spots were found to be significantly more intense in GCF. They included superoxide dismutase 1 (SOD1), apolipoprotein A-I (ApoA-I), and dermcidin (DCD). Moreover, GCF proteins from healthy subjects were broken down into small peptide fragments and then analyzed directly by LC-MS/MS analysis. A total of 327 proteins including ApoA-I, SOD1, and DCD were identified in GCF. These results may serve as reference for future proteomic studies searching for GCF biomarkers of periodontal diseases.     

Identification of target proteins of N-acetylglucosaminyl-transferase V and fucosyltransferase 8 in human gastric tissues by glycomic approach

To understand better the mechanism underlying gastric cancer and search for potential markers for gastric cancer prognosis, the proteomic analysis of gastric cancer tissues was conducted using two-dimensional gel electrophoresis and lectin blot, followed by electrospray ionization-mass spectrometry. These approaches permitted identification of glyco- or putative glycosylated proteins which may be associated with tumorigenesis. The proteins identified include molecules involved in sugar metabolism, signal transduction, proteolysis, and stress, as well as several unknown proteins, which were aberrantly glycosylated as evidenced by the L-phytohemagglutinin blot.     

幽门螺杆菌适应性定植蒙古沙鼠前、后的蛋白质组学研究

为比较研究幽门螺杆菌(Hp)适应性定植蒙古沙鼠前后的蛋白表达谱变化,将Hp临床分离株感染沙鼠,并体内连续传代,驯化一株高适应性菌株,然后采用双向电泳和质谱技术对适应性变化前后两株菌的差异蛋白进行鉴定。结果表明,随着Hp临床株在沙鼠体内的连续传代,感染率逐渐升高,第10代后,感染率稳定在90%以上。适应性定植后,Hp蛋白谱发生了变化。在选择的5个候选差异蛋白点中,共鉴定出4个蛋白,分别为Hp菌的功能未知的HP0318编码蛋白、氢化酶表达/形成蛋白(hypB)、异柠檬酸脱氢酶(icd)、ADP-L-D-甘露庚糖表异构酶(rfaD)。上述鉴定蛋白可能与Hp适应性定植具有很大的相关性。     

Proteome analysis of recombinant Escherichia coli producing human glucagon-like peptide-1

The proteomic response of recombinant Escherichia coli producing human glucagon-like peptide-1 was analyzed by two-dimensional gel electrophoresis. Protein spots in two-dimensional gel could be identified by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and their expression profiles were compared with those of nonproducing cells. Thirty-five intracellular proteins exhibited differential expression levels between the production and control strains. These changes reflected physiological responses to heterologous peptide production in recombinant E. coli. Specifically, physiological changes included the down-regulation of proteins involved in the central carbon metabolism, biosynthesis of cellular building blocks and peptides, and up-regulation of cell protection proteins and some sugar transport proteins. This comprehensive analysis would provide useful information for understanding physiological alterations to heterologous peptide production and for designing efficient metabolic engineering strategies for the production of recombinant peptides in E. coli.     

Comparative proteomic analysis of Helicobacter pylori strains associated with iron deficiency anemia

Helicobacter pylori is known to cause chronic gastritis, peptic ulcer, and gastric cancer, and has also been linked to iron deficiency anemia (IDA). To determine whether H. pylori clinical isolates correlate with the prevalence of H. pylori-associated IDA, we compared the proteomic profiles of H. pylori strains isolated from antral biopsy specimens of H. pylori-positive patients with or without IDA. Fifteen strains, including eight non-IDA and seven IDA strains, were cultured under iron-rich and iron-depleted conditions and then analyzed for protein expression profiles by 2-DE. The distances between two H. pylori strains were determined on the basis of similarities between their expression patterns of 189 protein spots, and a phylogenetic tree was constructed. The results revealed that the IDA strains formed a cluster separate from that of six non-IDA strains, with two non-IDA strains between the clusters. H. pylori strain 26695 was located in the non-IDA cluster. Protein spots displaying similar expression patterns were clustered, and 18 spots predominantly expressed in IDA strains were identified by MALDI-TOF analysis. These data indicate that the non-IDA and IDA strains can be distinguished by their protein expression profiles, suggesting that the polymorphism of H. pylori strains may be one of the factors determining the occurrence of H. pylori-associated IDA.