Proteomic approach to identify acute phase response-related proteins with low molecular weight in loach skin following injury

Proteome analysis by two-dimensional gel electrophoresis (2-DE) together with mass spectrometry was applied to screen acute phase response (APR)-related proteins with low molecular weight in loach skin following injury. Furthermore, Western blotting and function tests were applied to confirm the results obtained from the proteomic study. Fifteen APR-related proteins with sixteen spots (PLA with two spots) on a 2-DE map were identified in this study. Furthermore, six were known acute phase proteins including galactose-binding lectin (GBL), lysozyme, C3, CD59, double PLA and 50s ribosomal protein; while ATP kinase, zinc finger protein 183, alpha-neurotoxin homology, angiostatin, serine/threonine kinase, metalloproteinase inhibitor, regulator of G-protein 4, cryptdin-9 and disintegrin trigranin were found by our lab to be APR-related proteins. In addition, our results suggest that proteomes with low molecular weight can be characterized by 2-DE with a Tris-tricine system followed by mass spectrometry.     

Structural proteome of human colostral fat globule membrane proteins

Milk fat globule membrane (MFGM) contains proteins derived from the apical membrane of secreting epithelial cells of the mammary gland. Between 2-4% of total human milk protein content is associated with the fat globule fraction, as MFGM proteins. While MFGM proteins have very low classical nutritional value, they play important roles in various cell processes and defence mechanisms for the newborn. To date, fewer than 30 human MFGM proteins have been identified and characterized, either by immunological methods or by Edman sequencing and mass spectrometry. This study aimed to update the structural proteome of human colostral MFGM proteins and to create an annotated two-dimensional electrophoresis (2-DE) MFGM protein database available on-line. More than one hundred 2-DE spots derived from human colostral MFGM proteins were investigated by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and proteins were identified by three different software packages available on the web (PeptIdent, MS-Fit and ProFound); uncertain identifications were solved by nanoelectrospray ionization-ion trap mass spectrometry using SEQUEST software.     

Proteomic analysis of amniotic membrane prepared for human transplantation: characterization of proteins and clinical implications

Amniotic membrane is commonly exploited in several surgical procedures. Despite a freeze preservation period, it is reported to retain wound healing, anti-angiogenic, antiinflammatory and anti-scarring properties; however, little is known about the active protein content. 2-DE analysis of transplant-ready amniotic membrane (TRAM) was performed. The effects of preservation and processing on amnion proteome were investigated, and the major proteins in the TRAM characterized using mass spectrometry and immunoblotting. This identified a spectrum of proteins including thrombospondin, mimecan, BIG-H3, and integrin alpha 6. Preservation compromises cellular viability resulting in selective elution of soluble cellular proteins, leaving behind extracellular matrix-associated and cell structural proteins. A number of key architectural proteins common to the architecture of the ocular surface were demonstrated in AM, which are involved in homeostasis and wound healing. Handling procedures alter the protein composition of amniotic membrane prepared for transplantation. Without standardization, there will be inter-membrane variation, which may compromise the desired therapeutic effect of transplant ready amniotic membrane.     

Comparative proteome analysis of Helicobacter pylori

Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2-DE patterns with their identified protein species were added to the dynamic 2D-PAGE database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.     

Comparative proteomics analysis of serum proteins in ulcerative colitis patients

In the present study, we investigated the differentially expressed proteins associated with ulcerative colitis (UC) using proteomic methods. Two-dimensional electrophoresis (2-DE) technology was performed to separate the total proteins of ulcerative tissues from those of the normal tissues of UC patients. PDQuest software was applied to analyze the obtained 2-DE images. Candidate protein spots between the two groups were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and bioinformatics analysis. The well resolution and reproducible 2-DE patterns of UC and normal tissues were established. Of the 12 differentially expressed proteins, 9 were successfully identified, of which 6 proteins were up-regulated including apolipoprotein C-III, haptoglobin, receptor tyrosine kinase, aldehyde reductase, pericentriolar material 1, and heat shock factor protein 2, and 3 were down-regulated including keratin, filamin A-interacting protein 1, and tropomyosin 3. These identified proteins were related to hormonal modulation, immune response, oxidative stress, and signal conduction. The 2-DE protein expression profile of the UC tissues displays an obvious difference from that of the normal controls. Various proteins may be involved in the occurrence of UC.     

Identification of proteins induced by polycyclic aromatic hydrocarbon in Mycobacterium vanbaalenii PYR-1 using two-dimensional polyacrylamide gel electrophoresis and de novo sequencing methods

Protein profiles of Mycobacterium vanbaalenii PYR-1 grown in the presence of high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) were examined by two-dimensional gel electrophoresis (2-DE). Cultures of M. vanbaalenii PYR-1 were incubated with pyrene, pyrene-4,5-quinone (PQ), phenanthrene, anthracene, and fluoranthene. Soluble cellular protein fractions were analyzed and compared, using immobilized pH gradient (IPG) strips. More than 1000 gel-separated proteins were detected using a 2-DE analysis program within the window of isoelectric point (pI) 4-7 and a molecular mass range of 10-100 kDa. We observed variations in the protein composition showing the upregulation of multiple proteins for the five PAH treatments compared with the uninduced control sample. By N-terminal sequencing or mass spectrometry, we further analyzed the proteins separated by 2-DE. Due to the lack of genome sequence information for this species, protein identification provided an analytical challenge. Several PAH-induced proteins were identified including a catalase-peroxidase, a putative monooxygenase, a dioxygenase small subunit, a small subunit of naphthalene-inducible dioxygenase, and aldehyde dehydrogenase. We also identified proteins related to carbohydrate metabolism (enolase, 6-phosphogluconate dehydrogenase, indole-3-glycerol phosphate synthase, and fumarase), DNA translation (probable elongation factor Tsf), heat shock proteins, and energy production (ATP synthase). Many proteins from M. vanbaalenii PYR-1 showed similarity with protein sequences from M. tuberculosis and M. leprae. Some proteins were detected uniquely upon exposure to a specific PAH whereas others were common to more than one PAH, which indicates that induction triggers not only specific responses but a common response in this strain.     

脑出血与脑缺血动物模型脾淋巴细胞的蛋白质组学研究

目的：通过比较正常与脑出血及脑缺血模型大鼠脾淋巴细胞蛋白质表达的差异,初步探讨细胞免疫功能与脑血管病之间的关系。方法：将SD大鼠随机分为正常组、脑出血模型组（采用VII型胶原酶诱导脑出血）和局灶性脑缺血模型组（采用线栓法造成大脑中动脉阻塞）,分离大鼠脾淋巴细胞,提取总蛋白质后进行双向凝胶电泳,考马斯亮蓝染色,PDQUEST软件分析,对差异蛋白质点采用基质辅助激光解析电离质谱（MALDI-TOF-MS）技术进行鉴定并分析。结果：胶质细胞成熟因子 等9个蛋白在脑出血和脑缺血模型组表达上调,膜联蛋白III在脑出血和脑缺血模型组表达下调。结论：建立了分辨率高重复性较好的脑出血及局灶性脑缺血脾淋巴细胞总蛋白的双向凝胶电泳图谱,并鉴定一些与脑血管病脑损伤相关的差异表达蛋白质,为深入研究脑血管病细胞免疫功能改变与脑血管病之间的关系奠定了基础。     

橡胶树死皮病胶乳C-乳清差异表达蛋白质的筛选与鉴定

橡胶树死皮病(Tapping Panel Dryness,TPD)在世界各橡胶种植园普遍发生,给橡胶种植业带来严重的危害。为了更好地了解和阐明死皮病发生、发展的分子机制,本研究应用双向凝胶电泳技术（2-DE）比较橡胶树死皮株与健康株胶乳C-乳清蛋白质组表达的差异。采用固相pH梯度双向凝胶电泳分离橡胶树死皮株与健康株C-乳清的总蛋白质,凝胶经考染显色后,用PDQuest7.40图像分析软件进行比较分析,识别差异表达的蛋白质。这些点经过胶内酶切后进行基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)分析获取肽质指纹图谱(PMF),Mascot软件搜索SWISS-PROT和NCBInr数据库鉴定蛋白质。结果：①橡胶树死皮株与健康株C-乳清凝胶的平均蛋白质点数分别为1075±35和1134±27,其平均匹配的点数分别为982±38和1008±22,组内图像匹配率达91.89﹪和88.72﹪。②橡胶树死皮株与健康株C-乳清组间的平均匹配蛋白点数为970±25。利用MALDI-TOF-MS质谱技术对40个差异明显的蛋白点进行分析鉴定,通过查询数据库鉴定了27个蛋白质。本研究建立了分辨率高且重复性较好的橡胶树死皮株与 健康株胶乳C-乳清的双向凝胶电泳图谱,并应用质谱技术鉴定了其中表达差异的蛋白质点,这些差异表达的蛋白质可能参与了死皮发生和发展的过程。     

A reference map of a human pituitary adenoma proteome

In order to compare the proteomes from different cell types of pituitary adenomas for our long-term goal to clarify the molecular mechanisms that participate in the formation of pituitary adenoma, and to detect any tumor-related marker for an "early-stage" diagnosis, the two-dimensional gel electrophoresis (2-DE) reference map of a pituitary adenoma tissue proteome is described here. A vertical, two-dimensional (2-D) polyacrylamide gel electrophoresis system and PDQuest image analysis software have been used to provide a high level of between-gel reproducibility and to accurately array each protein expressed in a pituitary adenoma tissue. Mass spectrometry (matrix-assisted laser desorption/ionization-time of flight MALDI-TOF and liquid chromatography-electrospray ionization-quadrupole-ion trap LC-ESI-Q-IT) and protein databases were used to characterize each protein in the 2-D gel. The results demonstrate that a good reproducibility of the 2-D gel pattern was attained. The position deviation of matched spots among four 2-D gels was 1.95 +/- 0.45 mm in the isoelectric focusing direction, and 1.70 +/- 0.53 mm in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis direction. A total of ca. 1000 protein spots were separated by 2-DE, and 135 protein spots that represent 111 proteins were characterized with mass spectrometry (96 spots for MALDI-TOF, 39 spots for LC-ESI-Q-IT). The characterized proteins include pituitary hormones, cellular signals, enzymes, cellular-defense proteins, cell-structure proteins, transport proteins, etc. Those proteins were located in the cytoplasmic, cellular membrane, mitochondrial, endoplasmic reticulum, nuclear, ribonucleosome, extracellular fractions, or were secreted in plasma, etc. Those identified proteins contribute to a functional profile of the pituitary adenoma proteome. These data will be used to expand the proteome database of the human pituitary, which can be accessed in the website http://www.utmem.edu /proteomics.     

Towards the proteome of the marine bacterium Rhodopirellula baltica: mapping the soluble proteins

The marine bacterium Rhodopirellula baltica, a member of the phylum Planctomycetes, has distinct morphological properties and contributes to remineralization of biomass in the natural environment. On the basis of its recently determined complete genome we investigated its proteome by 2-DE and established a reference 2-DE gel for the soluble protein fraction. Approximately 1000 protein spots were excised from a colloidal Coomassie-stained gel (pH 4-7), analyzed by MALDI-MS and identified by PMF. The non-redundant data set contained 626 distinct protein spots, corresponding to 558 different genes. The identified proteins were classified into role categories according to their predicted functions. The experimentally determined and the theoretically predicted proteomes were compared. Proteins, which were most abundant in 2-DE gels and the coding genes of which were also predicted to be highly expressed, could be linked mainly to housekeeping functions in glycolysis, tricarboxic acid cycle, amino acid biosynthesis, protein quality control and translation. Absence of predictable signal peptides indicated a localization of these proteins in the intracellular compartment, the pirellulosome. Among the identified proteins, 146 contained a predicted signal peptide suggesting their translocation. Some proteins were detected in more than one spot on the gel, indicating post-translational modification. In addition to identifying proteins present in the published sequence database for R. baltica, an alternative approach was used, in which the mass spectrometric data was searched against a maximal ORF set, allowing the identification of four previously unpredicted ORFs. The 2-DE reference map presented here will serve as framework for further experiments to study differential gene expression of R. baltica in response to external stimuli or cellular development and compartmentalization.     

Characterisation of host defence proteins in milk using a proteomic approach

Besides providing nutrition to the newborn, milk also protects the neonate and the mammary gland against infection. As well as the six major proteins, bovine milk contains minor proteins, not all of which have been characterized. In this study, we have subjected bovine skim milk, whey, and milk fat globule membrane (MFGM) fractions to both direct liquid chromatography-tandem mass spectrometry (LC-MS/MS), and two-dimensional electrophoresis (2-DE) followed by matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) of individual protein spots to better characterize the repertoire of minor milk proteins, particularly those involved with host defense. Milk from peak lactation as well as during the period of colostrum formation and during mastitis were analyzed to gain a more complete sampling of the milk proteome. In total, 2903 peptides were detected by LC-MS and 2770 protein spots by 2-DE. From these, 95 distinct gene products were identified, comprising 53 identified through direct LC-MS/MS and 57 through 2-DE-MS. The latter were derived from a total of 363 spots analyzed with 181 being successfully identified. At least 15 proteins were identified that are involved in host defense. These results demonstrate that the proteome of milk is more complex than has previously been reported and a significant fraction of minor milk proteins are involved in protection against infection.     

The serum proteome of Equus caballus

We constructed a reference two-dimensional protein map for horse (Equus caballus) serum. The serum proteins were separated by two-dimensional electrophoresis (2-DE); 29 different gene products were identified. Proteins represented by 25 spots/spot groups were identified by tandem nanoelectrospray mass spectrometry (MS), four by matrix-assisted laser desorption ionization time-of-flight (TOF) MS and one was sequenced by TOF-TOF technology. The identities of four proteins were deduced by similarity to the human plasma protein database. In selected cases, i.e. the immunoglobulins, immunoblotting with specific antibodies provided additional information about the respective proteins. Albumin was detected as the full-length protein and as fragments of various sizes. Spots representing products of different mass and charge were also detected for alpha1-antitrypsin, haptoglobin and transthyretin. Thus, despite the fact that the Equus caballus genome is incompletely characterized, we were able to identify almost all moderate to high abundance proteins stained in the serum 2-DE pattern.     

An iterative calibration method with prediction of post-translational modifications for the construction of a two-dimensional electrophoresis database of mouse mammary gland proteins

Protein databases serve as general reference resources providing an orientation on two-dimensional electrophoresis (2-DE) patterns of interest. The intention behind constructing a 2-DE database of the water soluble proteins from wild-type mouse mammary gland tissue was to create a reference before going on to investigate cancer-associated protein variations. This database shall be deemed to be a model system for mouse tissue, which is open for transgenic or knockout experiments. Proteins were separated and characterized in terms of their molecular weight (M(r)) and isoelectric point (pI) by high resolution 2-DE. The proteins were identified using prevalent proteomics methods. One method was peptide mass fingerprinting by matrix-assisted laser desorption/ionization-mass spectrometry. Another method was N-terminal sequencing by Edman degradation. By N-terminal sequencing M(r) and pI values were specified more accurately and so the calibration of the master gel was obtained more systematically and exactly. This permits the prediction of possible post-translational modifications of some proteins. The mouse mammary gland 2-DE protein database created presently contains 66 identified protein spots, which are clickable on the gel pattern. This relational database is accessible on the WWW under the URL: http://www.mpiib-berlin.mpg.de/2D-PAGE.     

Detection of activity and mass spectrometric identification of mouse liver carboxylesterase and aldehyde dehydrogenase separated by non-denaturing two-dimensional electrophoresis after extraction with detergents

To examine the activities and identity of enzymes associated with organelles such as microsomes and mitochondria, proteins from mouse liver were extracted using the non-ionic detergents Nonidet P-40 (NP-40), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene isooctylphenyl ester (Triton X), n-octyl beta-D-glucoside (octyl glycoside) or anionic detergent sodium dodecylsulfate (SDS) after the removal of cytosolic proteins. The proteins extracted by detergents were separated by non-denaturing two-dimensional electrophoresis (2-DE). The activities of esterase and aldehyde dehydrogenase were retained by non-denaturing 2-DE after treatment with each non-ionic detergent, but the activities were reduced or lost when the proteins were extracted with more than 0.5% SDS. For proteomic analysis of the organelle-associated proteins in mouse liver, proteins were separated by non-denaturing 2-DE and were identified using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after the proteins were solubilized by octyl glycoside, NP-40 and 0.1% SDS. Several organelle-associated proteins such as carboxylesterase, aldehyde dehydrogenase, glucose regulated protein and HSP60 were identified. These results indicate that the activities and identity of detergent-soluble enzymes can be examined by this non-denaturing 2-DE and mass spectrometry.     

Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional electrophoresis and isotope-coded affinity tag technology

Classical proteomics combined two-dimensional gel electrophoresis (2-DE) for the separation and quantification of proteins in a complex mixture with mass spectrometric identification of selected proteins. More recently, the combination of liquid chromatography (LC), stable isotope tagging, and tandem mass spectrometry (MS/MS) has emerged as an alternative quantitative proteomics technology. We have analyzed the proteome of Mycobacterium tuberculosis, a major human pathogen comprising about 4,000 genes, by (i) 2-DE and mass spectrometry (MS) and by (ii) the isotope-coded affinity tag (ICAT) reagent method and MS/MS. The data obtained by either technology were compared with respect to their selectivity for certain protein types and classes and with respect to the accuracy of quantification. Initial datasets of 60,000 peptide MS/MS spectra and 1,800 spots for the ICAT-LC/MS and 2-DE/MS methods, respectively, were reduced to 280 and 108 conclusively identified and quantified proteins, respectively. ICAT-LC/MS showed a clear bias for high M(r) proteins and was complemented by the 2-DE/MS method, which showed a preference for low M(r) proteins and also identified cysteine-free proteins that were transparent to the ICAT-LC/MS method. Relative quantification between two strains of the M. tuberculosis complex also revealed that the two technologies provide complementary quantitative information; whereas the ICAT-LC/MS method quantifies the sum of the protein species of one gene product, the 2-DE/MS method quantifies at the level of resolved protein species, including post-translationally modified and processed polypeptides. Our data indicate that different proteomic technologies applied to the same sample provide complementary types of information that contribute to a more complete understanding of the biological system studied.     

Protein expression by Streptococcus mutans during initial stage of biofilm formation

Cells growing on surfaces in biofilms exhibit properties distinct from those of planktonic cells, such as increased resistance to biocides and antimicrobial agents. In spite of increased interest in biofilms, very little is known about alterations in cell physiology that occur upon attachment of cells to a surface. In this study we have investigated the changes induced in the protein synthesis by contact of Streptococcus mutans with a surface. Log-phase planktonic cells of S. mutans were allowed to adhere to a glass slide for 2 h in the presence of a (14)C-amino acid mixture. Nonadhered cells were washed away, and the adhered cells were removed by sonication. The proteins were extracted from the nonadhered planktonic and the adhered biofilm cells and separated by two-dimensional gel electrophoresis followed by autoradiography and image analysis. Image analysis revealed that the relative rate of synthesis of 25 proteins was enhanced and that of 8 proteins was diminished > or =1.3-fold in the biofilm cells. Proteins of interest were identified by mass spectrometry and computer-assisted protein sequence analysis. Of the 33 proteins associated with the adhesion response, all but 10 were identified by mass spectrometry and peptide mass fingerprinting. The most prominent change in adhered cells was the increase in relative synthesis of enzymes involved in carbohydrate catabolism indicating that a redirection in protein synthesis towards energy generation is an early response to contact with and adhesion to a surface.     

Enrichment and proteome analysis of a hyperthermostable protein set of archaeon Thermococcus onnurineus NA1

Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that can be used for the screening of thermophilic enzymes. Previously, we characterized the metabolic enzymes of the cytosolic proteome by two-dimensional electrophoresis/tandem mass spectrometry (2-DE/MS-MS). In this study, we identified a subset of hyperthermostable proteins in the cytosolic proteome using enrichment by in vitro heat treatment and protein identification. After heat treatment at 100°C for 2 h, 13 and 149 proteins were identified from the soluble proteome subset by 2-DE/MS-MS and 1-DE/MS-MS analysis, respectively. Representative proteins included intracellular protease I, thioredoxin reductase, triosephosphate isomerase, putative hydroperoxide reductase, proteasome, and translation initiation factors. Intracellular protease, deblocking aminopeptidases, and fructose-1,6-bisphosphatase were overexpressed in Escherichia coli and biological activity above 85°C was confirmed. The folding transition temperature (Tm) of identified proteins was analyzed using the in silico prediction program TargetStar. The proteins enriched with the heat treatment have higher Tm than the homologous proteins from mesophilic strains. These results suggested that the heat-stable protein set of hyperthermophilic T. onnurineus NA1 can be effectively fractionated and enriched by in vitro heat treatment.