Mapping and comprehensive analysis of the extracellular and cell surface proteome of the human pathogen Corynebacterium diphtheriae

Secreted proteins of the human pathogen Corynebacterium diphtheriae might be involved in important pathogen-host cell interactions. Here, we present the first systematic reference map of the extracellular and cell surface proteome fractions of the type strain C. diphtheriae C7s(-)tox-. The analysis window of 2-DE covered the pI range from 3 to 10 along with a MW range from 8 to 150 kDa. Computational analysis of the 2-D gels detected almost 150 protein spots in the extracellular proteome fraction and about 80 protein spots of the cell surface proteome. MALDI-TOF-MS and PMF with trypsin unambiguously identified 107 extracellular protein spots and 53 protein spots of the cell surface, representing in total 85 different proteins of C. diphtheriae C7s(-)tox-. Several of the identified proteins are encoded by pathogenicity islands and might represent virulence factors of C. diphtheriae. Additionally, four solute-binding proteins (HmuT, Irp6A, CiuA, and FrgD) of different iron ABC transporters were identified, with the hitherto uncharacterized FrgD protein being the most abundant one of the cell surface proteome of C. diphtheriae C7s(-)tox-.     

Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).     

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.     

红海榄根部盐胁迫反应的比较蛋白质组学分析

红海榄(Rhizophora stylosa)是一种典型的红树林盐生植物.本研究利用蛋白组学技术对淡栽(R0)和3% NaCl盐栽(R3)处理后的红海榄根部总蛋白进行了比较研究.双向电泳图谱的结果表明,R0和R3分别有981和972个蛋白点,蛋白点主要集中在分子量28～70 kD,等电点4.0～8.5之间. R0和R3之间差异明显的有15个蛋白点.其中,8个蛋白的表达量在R0中表达增高（10倍）,而在R3中相对下降.另外,7个蛋白的表达量在R0中较低,而在R3中表达量显著增高.对这15个蛋白点进行肽质量指纹图谱分析,10个蛋白点找到匹配蛋白.功能预测分析发现,在盐水栽培上调的蛋白质一般与逆境胁迫有关,淡水栽培上调的蛋白质一般与基本代谢有关.这些研究结果为进一步研究红海榄的耐盐机理提供了有意义的线索.     

Proteomic dataset of Sca-1+ progenitor cells

Embryonic stem cells (ES cells) can differentiate into endothelial cells and smooth muscle cells (SMCs), which participate in vascular angiogenesis. In this study, we differentiated mouse ES cells into Sca-1(+) cells, which have the potential to serve as vascular progenitor cells, and mapped their proteome by 2-DE using a pH 3-10 non-linear gradient and 12% SDS-polyacrylamide gels. A subset of 300 protein spots was analysed and mapped, with 241 protein spots being identified by their PMF using MALDI-TOF MS or by partial amino acid sequencing using MS/MS. Our protein map is the first of Sca-1(+) progenitor cells and will facilitate the identification of proteins differentially expressed during stem cell differentiation. The proteome of adult arterial SMCs is described in an accompanying paper (in this issue, DOI 10.1002/pmic.200402045). All data are made accessible on our website http://www.vascular-proteomics.com.     

应用优化的双向电泳技术建立纤维堆囊菌So0157-2蛋白质组数据库

堆囊菌丰富的次级代谢产物是新药的重要来源,而蛋白质组学分析是研究代谢调控的有效方法．然而堆囊菌含有大量的胞外多糖以及黏液,干扰了蛋白质组学分析中蛋白质的溶解度、分辨率及重现性．为了高通量地筛选Sorangium cellulosum So0157-2表达的特异性蛋白,实验优化了S. cellulosum So0157-2双向电泳方法．首先,S. cellulosum So0157-2蛋白在裂解液中有更好的溶解度．pH 3～10非线性胶条和1 mg的蛋白上样量适用于第一向等电聚焦,分别提高了蛋白质点的分辨率和低丰度蛋白质的表达．15% SDS-PAGE 改善了S. cellulosum So0157-2蛋白分离的分辨率和重现性．最终,通过优化的双向电泳方法获得了S. cellulosum So0157-2 在M26培养基中培养3天的全蛋白质表达谱,并检测到552个蛋白质点．进而对表达蛋白通过MALDI-TOF-MS进行质谱鉴定,其中474个蛋白质得到鉴定,鉴定率85.9%．得到鉴定的蛋白质包括细胞结构和功能组分,以及细胞代谢合成酶类,其中8个蛋白质与糖类的转化和代谢相关,这有助于糖基化埃博霉素A的深入研究．该优化方法为进一步建立纤维堆囊菌So0157-2在各种培养条件下的蛋白质组表达数据库打下基础．     

Proteomic analysis of glutamine-treated human intestinal epithelial HCT-8 cells under basal and inflammatory conditions

Glutamine (Gln) promotes intestinal growth and maintains gut structure and function, especially in situations of injury and during inflammation. Several mechanisms could contribute to Gln protective effects on gut. Proteomics enable us to characterize differentially expressed proteins in tissues in response to modifications of the biological or nutritional environment. Gln effects on the human intestinal epithelial HCT-8 cell line proteome were assessed under basal and proinflammatory conditions. The 2-DE gels were obtained and compared. Proteins were identified by MS and using databases. About 1200 spots were detected in both 2- and 10-mM Gln concentrations. Under basal conditions, 24 proteins were differentially expressed in response to Gln. Half of these proteins were implicated in protein biosynthesis or proteolysis and 20% in membrane trafficking. Under proinflammatory conditions, 27 proteins were up- or down-regulated by Gln 10 mM. From these proteins, 40% were involved in protein biosynthesis or proteolysis, 16% in membrane trafficking, 8% in cell cycle and apoptosis mechanisms and 8% in nucleic acid metabolism. This study provides the first holistic picture of proteome modulation by Gln in a human enterocytic cell line under basal and proinflammatory conditions, and supports further evaluation of nutritional modulation of intestinal proteome in humans.     

食源性肥胖大鼠下丘脑差异表达蛋白的蛋白质组学分析

建立食源性肥胖大鼠模型,对正常大鼠和肥胖大鼠下丘脑全蛋白进行双向凝胶电泳,产生下丘脑蛋白双向凝胶电泳图谱.对图谱进行比对分析后,从凝胶上切取差异表达的蛋白点,经胶内酶解,通过基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS) 对酶解后的肽段进行分析,再经数据库（NCBInr）检索,对蛋白质进行鉴定.研究发现,正常组表达图谱可检测到1　160±15（n=5）个蛋白点,肥胖组表达图谱可检测到1　070±10 （n=5）个蛋白点,与对照组相比,匹配率大于80％.并且成功鉴定了17种差异表达蛋白质,其中有7 种在肥胖组表达上调,10种表达下调.它们分别属于代谢酶、细胞周期调控因子、抗氧化蛋白、信号传导蛋白、蛋白酶体相关蛋白、细胞骨架蛋白以及未知蛋白等. 与正常对照组相比,肥胖组的蛋白质表达存在着较大差异,通过对差异表达蛋白的分析,提示了在肥胖发生的过程中,下丘脑神经中枢经历了一个非常复杂的信号活动和特定改变,为深入认识肥胖的发病机制奠定了基础.     

Quantitative proteome analysis of CD95 (Fas/Apo-1)-induced apoptosis by stable isotope labeling with amino acids in cell culture, 2-DE and MALDI-MS

Proteome analysis of Jurkat T cells induced to undergo apoptosis by CD95 (Fas/Apo-1) treatment was performed to identify modified proteins. We used stable isotope labeling with amino acids in cell culture (SILAC) using leucine to identify proteins of apoptotic and control Jurkat T cells by 2-DE and MALDI-MS. Out of 224 spots analyzed, we quantified 213 spots with 3.5 leucine-containing peptide pairs on average; 28 proteins with a relative abundance of higher than 1.5 were found. Five new modified proteins including calcyclin binding protein, cytosolic acyl coenzyme A thioester hydrolase, heterogeneous ribonucleoprotein M, replication factor C 37-kDa subunit, and tropomyosin 4 chain were identified as being modified in response to apoptosis. In comparison to differential proteome analysis via silver-stained 2-D gels and PMF of total Jurkat T cell lysates, 15 additional apoptosis-modified proteins were identified though 8 proteins were not found. The described approach using SILAC instead of silver staining for relative quantification was simpler to perform regarding the number of required 2-D gels, that cumbersome gel comparisons were avoided, and more apoptosis-modified proteins were identified, but with a higher demand on data interpretation of the mass spectra obtained.     

Proteomic Analysis for the Identification of Proteins Related to Methotrexate Resistance

Methotrexate(MTX) is one of the most important and frequently used drugs in cancer therapy, but the efficacy of this drug is often compromised by the development of resistance in cancer cells. To seek and identify differentially expressed proteins related to MTX resistance and provide clues for the mechanism of MTX resistance, proteins from cell line MTX300 (resistant to 300 μmol/L MTX) and its control cell line 3T3R500 were separated by two-dimensional electrophoresis (2-DE). The colloidal Coomassie brilliant blue-stained 2-DE gels were subjected to image analysis, which revealed several spots with high levels of differential expression between MTX300 and 3T3R500. The protein spot with highest differential expression was submitted for tryptic peptide mass fingerprinting(PMF) for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MS analysis and database searches revealed it to be dihydrofolate reductase (DHFR), which was subsequently confirmed by Western blot. The result suggested that DHFR might play an important role in the MTX resistance.     

运用蛋白质组学技术研究与氨甲蝶呤耐药性相关的蛋白质

氨甲蝶呤（MTX）是一种重要的常用化疗药物,然而由于肿瘤细胞对其耐药性的增强而经常导致其疗效大大降低。为了寻找并鉴定与MTX耐药性相关的蛋白质从而为进一步闸明MTX的耐药机制提供线索,培养来源于小鼠NIH3T3的小鼠胚胎成纤维细胞系3T3R500与其耐300μmol／L MTX的细胞系MTX300,提取上述两种细胞系的总蛋白质,双向凝胶电泳分离蛋白质组成分,扫描并通过软件分析考马斯亮蓝染色的2-DE凝胶,选取表达差异最显著的点,胶内酶切后MALDI-TOF-MS进行肽指纹图谱（PMF）鉴定。图像分析显示,实验组和对照组的蛋白质组图谱之间,一些蛋白质点的表达有明显的变化。通过MALDI-TOF-MS和数据库查询,成功鉴定了耐药后表达变化最显著的蛋白质点为二氢叶酸还原酶（DHFR）,并通过Western blot验证了该结果,提示DHFR在MTX耐药机制中发挥重要作用。     

Proteomic Screening of Antigenic Proteins from the Hard Tick,Haemaphysalis longicornis (Acari: Ixodidae)

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.     

Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.     

2-DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM)

The proteome of a proliferating human stem cell line was analyzed and then utilized to detect stem cell differentiation-associated changes in the protein profile. The analysis was conducted with a stable human fetal midbrain stem cell line (ReNcell VM) that displays the properties of a neural stem cell. Therefore, acquisition of proteomic data should be representative of cultured human neural stem cells (hNSCs) in general. Here we present a 2-DE protein-map of this cell line with annotations of 402 spots representing 318 unique proteins identified by MS. The subsequent proteome profiling of differentiating cells of this stem cell line at days 0, 4 and 7 of differentiation revealed changes in the expression of 49 identified spots that could be annotated to 45 distinct proteins. This differentiation-associated expression pattern was validated by Western blot analysis for transgelin-2, proliferating cell nuclear antigen, as well as peroxiredoxin 1 and 4. The group of regulated proteins also included NudC, ubiquilin-1, STRAP, stress-70 protein, creatine kinase B, glial fibrillary acidic protein and vimentin. Our results reflect the large rearrangement of the proteome during the differentiation process of the stem cells to terminally differentiated neurons and offer the possibility for further characterization of specific targets driving the stem cell differentiation.     

Proteomic analysis of cellular protein alterations using a hepatitis B virus-producing cellular model

Hepatitis B virus (HBV) is one of the major etiological factors responsible for acute and chronic liver disease and for the development of hepatocellular carcinoma (HCC). To determine the effects of HBV replication on host cell-protein expression, we utilized 2-DE and MS/MS analysis to compare and identify differentially expressed proteins between an HBV-producing cell line HepG2.2.15 and its parental cell line HepG2. Of the 66 spots identified as differentially expressed (+/- over twofold, p <0.05) between the two cell lines, 62 spots (corresponding to 61 unique proteins) were positively identified by MS/MS analysis. These proteins could be clearly divided into three major groups by cluster and metabolic/signaling pathway analysis: proteins involved in retinol metabolism pathway, calcium ion-binding proteins, and proteins associated with protein degradation pathways. Other proteins identified include those that function in diverse biological processes such as signal transduction, immune regulation, molecular chaperone, electron transport/redox regulation, cell proliferation/differentiation, and mRNA splicing. In summary, we profiled proteome alterations between HepG2.2.15 and HepG2 cells. The proteins identified in this study would be useful in revealing the mechanisms underlying HBV-host cell interactions and the development of HCC. This study can also provide some useful clues for antiviral research.     

Mapping of alkaline proteins in bovine skeletal muscle

In agricultural sciences, proteomics has become the new hope for analyzing the meat quality traits that are closely related to the skeletal muscle traits. 2-DE muscle maps of many species have been recently reported and used to find molecular markers of meat quality traits. However, one limitation of 2-DE based analyses is due to the limited alkaline protein separation. Considering this problem, there is a need to use recent advances that have markedly improved the 2-DE based analysis of alkaline proteins. Hence, the present study provides additional information concerning the alkaline proteome of bovine skeletal muscle by using an appropriate protocol to characterize proteins over the entire range of pH 7-11. A total of 32 distinct gene products corresponding to 60 protein spots were identified by PMF and grouped in seven categories according to their main function. This 2-D map will contribute to muscle proteome studies since a significant portion of proteins is in the alkaline pH range.