Proteomic Profiling of Mouse Helper T Cell Differentiation

Helper T cell differentiation is a key process in the regulation of adaptive immune responses. Here, mouse Th1 and Th2 cells are profiled using high‐throughput proteomics to increase the understanding of the molecular biology of Th differentiation to support the design of prophylactic and therapeutic intervention strategies for (infectious) diseases. Protein profiling of Th1/Th2 differentiated cells results in the quantification of almost 6000 proteins of which 41 are differentially expressed at FDR < 0.1, and 19 at the FDR < 0.05 level, respectively. Differential protein expression analysis identifies a number of the expected canonical Th differentiation markers, and gene set analysis using the REACTOME database and a hypergeometric test (FDR < 0.05) confirms that helper T cell pathways are the top sets that are differentially expressed. Additionally, by network analysis, many differentially expressed proteins are associated with the Th1 and Th2 pathways. Data are available via PRIDE database with identifier PXD004532.     

Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions

Identification of cytosolic proteins differentially expressed between types of leukemia and lymphoma may provide a molecular basis for classification and understanding their cellular properties. Two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry have been used to identify proteins that are differentially expressed in cytosolic extracts from four human leukemia and lymphoma cell lines: HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia) and Raji (B-cell Burkitt's lymphoma). A total of 247 differentially expressed proteins were identified between the four cell lines. Analysis of the data by principal component analysis identified 22 protein spots (17 different protein species) differentially expressed at more than a 95% variance level between these cell lines. Several of these proteins were differentially expressed in only one cell line: HL-60 (myeloperoxidase, phosphoprotein 32 family member A, ras related protein Rab-11B, protein disulfide-isomerase, ran-specific GTPase-activating protein, nucleophosmin and S-100 calcium binding protein A4), and Raji (ezrin). Several of these proteins were differentially expressed in two cell lines: Raji and MEC1 (C-1-tetrahydrofolate synthase, elongation factor 2, alpha- and beta-tubulin, transgelin-2 and stathmin). MEC1 and CCRF-CEM (gamma-enolase), HL-60 and CCRF-CEM (ubiquitin-conjugating enzyme E2 N). The differentially expressed proteins identified in these four cell lines correlate with cellular properties and provide insights into the molecular basis of these malignancies.     

Hsp 27 is better associated with the expression of inducible thermotolerance in human pancreatic tumor cell lines than hsp 70, p53 or p21/waf1/cip1

We evaluated the levels of p53, p21/waf1/cip1, hsp 27 and hsp 70 proteins in confluent cultures of human pancreatic tumor cell lines during the expression of inducible thermotolerance (IT). Two of these cell lines have wild type, whereas the other two have mutant, p53 status. Three cell lines expressed a significant amount of IT (p<0.05 and better, one-tailed Student's t-test); the fourth manifested little IT. Hsp 70 was upregulated in all cell lines. In contrast, hsp 27 was upregulated only in the cell lines that expressed IT. There was no correlation between either p53, or p21, protein levels and propensity to express IT. We conclude that hsp 27 is better associated with the expression of IT than hsp 70 in these cell lines.     

应用蛋白质组学方法初步筛选与鼻咽癌放射敏感相关的蛋白

目的:通过筛选放射敏感性不同的鼻咽癌细胞中差异表达蛋白,以发现与鼻咽癌放射敏感相关的蛋白。方法:放射处理并结合流式细胞术检测及比较5-8F和6-10B细胞的放射敏感性。提取细胞总蛋白,进行双向凝胶电泳、MALDI-TOF肽质指纹图分析、质谱数据的蛋白质库搜寻鉴定。应用Western Blot检测细胞中蛋白质表达。应用免疫组织化学方法检测鼻咽癌组织中相关蛋白的表达。结果:双向凝胶电泳后对胶上的部分分辨较好的差异蛋白质点进行肽质谱指纹图分析和鉴定,在两种细胞中差异表达最为显著的蛋白质有9个。Western Blot证实CK19和P73在5-8F和6-10B表达与蛋白质组结果一致。P73在鼻咽癌放射敏感组和不敏感组中的表达阳性率分别为90%、57.5%,存在显著性差异。结论:放射敏感性不同的鼻咽癌细胞中存在一些差异表达蛋白,这些蛋白可能与鼻咽癌放射敏感性有关,其中P73可能成为放射敏感性预测的侯选标志物。     

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.     

LCM纯化的人支气管上皮癌变各阶段组织的定量蛋白质组学研究

为分析支气管上皮癌变进程中的差异表达蛋白质,筛选肺鳞癌早期诊断标志物,以人支气管上皮癌变各阶段组织为研究对象,先采用激光捕获显微切割技术(LCM) 纯化人正常支气管上皮组织、鳞状化生、不典型增生、原位癌、浸润性肺鳞癌组织,再用同位素标记相对和绝对定量 (iTRAQ) 技术结合二维液相色谱串联质谱（2D LC-MS/MS）鉴定支气管上皮癌变进程中各阶段的差异表达蛋白质。结果共鉴定了1036个蛋白质,筛选出102个与人支气管上皮癌变相关的差异蛋白质,在这些差异蛋白质中,有的在支气管上皮癌变过程中进行性上调,有的在支气管上皮癌变过程中进行性下调,有的呈阶段特异性改变;功能分析表明,这些差异蛋白质涉及代谢、细胞凋亡、增殖、分化、信号传导、转录、翻译、细胞粘附、免疫反应与发育等。Western blotting 及免疫组织化学技术验证了其中 2个差异蛋白(S100A9和 CKB) 的表达,证实了定量蛋白质组学结果的可靠性。研究结果提示：这些差异表达蛋白质与支气管上皮癌变相关,并可成为肺鳞癌的早期诊断标志物,进一步研究差异蛋白的生物学功能,将有助于阐明支气管上皮的癌变机制,从而为肺鳞癌的早期诊断与发病机制研究提供新思路。     

Differential protein expression on the cell surface of colorectal cancer cells associated to tumor metastasis

Progression to metastasis is the critical point in colorectal cancer (CRC) survival. However, the proteome associated to CRC metastasis is very poorly understood at the moment. In this study, we used stable isotope labeling by amino acids in cell culture to compare two CRC cell lines: KM12C and KM12SM, representing poorly versus highly metastatic potential, to find and quantify the differences in protein expression, mostly at the cell surface level. After biotinylation followed by affinity purification, membrane proteins were separated by SDS‐PAGE and analyzed using nanoflow LC‐ESI‐LTQ. A total of 291 membrane and membrane‐associated proteins were identified with a p value<0.01, from which 60 proteins were found to be differentially expressed by more than 1.5‐fold. We identified a number of cell signaling, CDs, integrins and other cell adhesion molecules (cadherin 17, junction plakoglobin (JUP)) among the most deregulated proteins. They were validated by Western blot, confocal microscopy and flow cytometry analysis. Immunohistochemical analysis of paired tumoral samples confirmed that these differentially expressed proteins were also altered in human tumoral tissues. A good correlation with a major abundance in late tumor stages was observed for JUP and 17‐β‐hydroxysteroid dehydrogenase type 8 (HSD17B8). Moreover, the combined increase in JUP, occludin and F11 receptor expression together with cadherin 17 expression could suggest a reversion to a more epithelial phenotype in highly metastatic cells. Relevant changes were observed also at the metabolic level in the pentose phosphate pathway and several amino acid transporters. In summary, the identified proteins provide us with a better understanding of the events involved in liver colonization and CRC metastasis.     

Comparative proteome profiling and functional analysis of chronic myelogenous leukemia cell lines

The aim of the present study was the molecular profiling of different Ph+ chronic myelogenous leukemia (CML) cell lines (LAMA84, K562, and KCL22) by a proteomic approach. By employing two-dimensional gel electrophoresis combined with mass spectrometry analysis, we have identified 191 protein spots corresponding to 142 different proteins. Among these, 63% were cancer-related proteins and 74% were described for the first time in leukemia cells. Multivariate analysis highlighted significant differences in the global proteomic profile of the three CML cell lines. In particular, the detailed analysis of 35 differentially expressed proteins revealed that LAMA84 cells preferentially expressed proteins associated with an invasive behavior, while K562 and KCL22 cells preferentially expressed proteins involved in drug resistance. These data demonstrate that these CML cell lines, although representing the same pathological phenotype, show characteristics in their protein expression profile that suggest different phenotypic leukemia subclasses. These data contribute a new potential characterization of the CML phenotype and may help to understand interpatient variability in the progression of disease and in the efficacy of a treatment.     

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.     

Protein expression profiling of breast cancer cells by dissociable antibody microarray (DAMA) staining

Dissociable antibody microarray (DAMA) staining is a technology that combines protein microarrays with traditional immunostaining techniques. It can simultaneously determine the expression and subcellular location of hundreds of proteins in cultured cells and tissue samples. We developed this technology and demonstrated its application in identifying potential biomarkers for breast cancer. We compared the expression profiles of 312 proteins among three normal breast cell lines and seven breast cancer cell lines and identified 10 differentially expressed proteins by the data analysis program DAMAPEP (DAMA protein expression profiling). Among those proteins, RAIDD, Rb p107, Rb p130, SRF, and Tyk2 were confirmed by Western blot and statistical analysis to have higher expression levels in breast cancer cells than in normal breast cells. These proteins could be potential biomarkers for the diagnosis of breast cancer.     

Signalling through cerebral cavernous malformation protein networks

Cerebral cavernous malformations (CCMs) are neurovascular abnormalities characterized by thin, leaky blood vessels resulting in lesions that predispose to haemorrhages, stroke, epilepsy and focal neurological deficits. CCMs arise due to loss-of-function mutations in genes encoding one of three CCM complex proteins, KRIT1, CCM2 or CCM3. These widely expressed, multi-functional adaptor proteins can assemble into a CCM protein complex and (either alone or in complex) modulate signalling pathways that influence cell adhesion, cell contractility, cytoskeletal reorganization and gene expression. Recent advances, including analysis of the structures and interactions of CCM proteins, have allowed substantial progress towards understanding the molecular bases for CCM protein function and how their disruption leads to disease. Here, we review current knowledge of CCM protein signalling with a focus on three pathways which have generated the most interest—the RhoA–ROCK, MEKK3–MEK5–ERK5–KLF2/4 and cell junctional signalling pathways—but also consider ICAP1-β1 integrin and cdc42 signalling. We discuss emerging links between these pathways and the processes that drive disease pathology and highlight important open questions—key among them is the role of subcellular localization in the control of CCM protein activity.     

Quantitative proteome analysis of breast cancer cell lines using 18O-labeling and an accurate mass and time tag strategy

Proteome comparison of cell lines derived from cancer and normal breast epithelium provide opportunities to identify differentially expressed proteins and pathways associated with specific phenotypes. We employed 16O/18O peptide labeling, FT-ICR MS, and an accurate mass and time (AMT) tag strategy to simultaneously compare the relative abundance of hundreds of proteins in non-cancer and cancer cell lines derived from breast tissue. A cell line reference panel allowed relative protein abundance comparisons among multiple cell lines and across multiple experiments. A peptide database generated from multidimensional LC separations and MS/MS analysis was used for subsequent AMT tag-based peptide identifications. This peptide database represented a total of 2299 proteins, including 514 that were quantified in five cell lines using the AMT tag and 16O/18O strategies. Eighty-six proteins showed at least a threefold protein abundance change between cancer and non-cancer cell lines. Hierarchical clustering of protein abundance ratios revealed that several groups of proteins were differentially expressed between the cancer cell lines.     

Correlation of zinc finger protein 2, a prognostic biomarker,with immune infiltrates in liver cancer

Purpose: The expression and clinical value of zinc finger protein 2 gene (ZIC2) in hepatocellular carcinoma (HCC) were analyzed by mining gene information from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases.Methods: Gene chip data sets were retrieved from GEO and TCGA and screened for differentially expressed genes in HCC. Gene expression profile interaction analysis (GEPIA) and Kaplan–Meier curves were used to analyze the relationship between differentially expressed genes (DEGs) and survival and prognosis in patients with HCC. Moreover, the Genecards database was used to extract ZIC2-related proteins and to analyze the physiological process of protein enrichment. Furthermore, the relationships between ZIC2 gene and tumor cell immune invasion and that between immune cell infiltration and the 5-year survival rate were studied using the tumor immune evaluation resource (TIMER) database.Results: Datasets from GEO and TCGA revealed that ZIC2 was differentially expressed in HCC tissues and normal tissues (P<0.05). High ZIC2 expression was associated with overall survival (OS) and progress-free survival in HCC patients. Overall, 25 ZIC2 related proteins, including Gli3, PRKDC, and rnf180 were identified and protein enrichment analysis indicated these were associated with four types of cell components, six types of cell functions, and eight types of biological processes. ZIC2 was positively correlated with immune infiltration cells in patients with HCC, and higher expression of ZIC2 mRNA CD4+T cells is associated with a better 5-year survival.Conclusion: ZIC2 gene may be used as an immune response marker in liver cancer to predict the prognosis of HCC.     

Identification of differential proteins in nasopharyngeal carcinoma cells with p53 silence by proteome analysis

Although mutation of p53 tumor-suppressor gene is rare in nasopharyngeal carcinoma (NPC), NPC has a high frequency of overexpression of p53 protein. There seem to be complex mechanisms of inactivation and stabilization of p53 in NPC. To detect proteins associated with the function of p53 in high throughout screening, we succeeded in establishing p53 knockdown human NPC CNE2 cell line (CNE2sip53) using stable RNA interference, and compared the proteomic changes between CNE2sip53 and control cell line CNE2/pSUPER using two-dimensional gel electrophoresis. Twenty-two differentially expressed proteins between the two cell lines were identified by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry, some of which are known to be associated with the p53 function (HSP27, hnRNP K, 14-3-3sigma, etc.), and others may be novel proteins associated with p53 function (eIF4B, TPT1, hnRNP H3, SFRS1 etc.). Furthermore, several differential proteins including HSP27, HSP70, GRP75 and GRP78 were verified as p53 interacting proteins in NPC by immunoprecipitation and Western blot analysis, and the suppression of HSP27 expression by HSP27 antisense oligonucleotides could decrease the p53 protein level. Our data suggest that these differential proteins may be associated with the function of p53 in NPC, and provide new clues to elucidate the mechanisms of inactivation and stabilization of p53 in NPC.     

A global view of protein expression in human cells,tissues, and organs

Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high‐resolution, immunohistochemistry‐based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced.     

Exposure to Cobalt Causes Transcriptomic and Proteomic Changes in Two Rat Liver Derived Cell Lines

Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies.     

Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines

ABSTRACT: BACKGROUND: The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. RESULTS: We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. CONCLUSIONS: Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.     

Comparative Proteomic Profiling of Pancreatic Ductal Adenocarcinoma Cell Lines

Pancreatic cancer is one of the most fatal cancers and is associated with limited diagnostic and therapeutic modalities. Currently, gemcitabine is the only effective drug and represents the preferred first-line treatment for chemotherapy. However, a high level of intrinsic or acquired resistance of pancreatic cancer to gemcitabine can contribute to the failure of gemcitabine treatment. To investigate the underlying molecular mechanisms for gemcitabine resistance in pancreatic cancer, we performed label-free quantification of protein expression in intrinsic gemcitabine-resistant and - sensitive human pancreatic adenocarcinoma cell lines using our improved proteomic strategy, combined with filter-aided sample preparation, single-shot liquid chromatography-mass spectrometry, enhanced spectral counting, and a statistical method based on a power law global error model. We identified 1931 proteins and quantified 787 differentially expressed proteins in the BxPC3, PANC-1, and HPDE cell lines. Bioinformatics analysis identified 15 epithelial to mesenchymal transition (EMT) markers and 13 EMT-related proteins that were closely associated with drug resistance were differentially expressed. Interestingly, 8 of these proteins were involved in glutathione and cysteine/methionine metabolism. These results suggest that proteins related to the EMT and glutathione metabolism play important roles in the development of intrinsic gemcitabine resistance by pancreatic cancer cell lines.     

Maize (Zea mays L.) seedling leaf nuclear proteome and differentially expressed proteins between a hybrid and its parental lines

To better understand the underlying molecular basis of leaf development in maize, a reference map of nuclear proteins in basal region of seedling leaf was established using a combination of 2DE and MALDI‐TOF‐MS. In total, 441 reproducible protein spots in nuclear proteome of maize leaf basal region were detected with silver staining in a pH range of 3–10, among which 203 spots corresponding to 163 different proteins were identified. As expected, proteins implicated in RNA and protein‐associated functions were overrepresented in nuclear proteome. Remarkably, a high percentage (10%) of proteins was identified to be involved in cell division and growth. In addition, comparative nuclear proteomic analysis in leaf basal region of highly heterotic hybrid Mo17/B73 and its parental lines was also performed and 52 of 445 (11.69%) detected protein spots were differentially expressed between the hybrid and its parental lines, among which 16 protein spots displayed nonadditively expressed pattern. These results indicated that hybridization between two parental lines can cause changes in the expression of a variety of nuclear proteins, which may be responsible for the observed leaf size heterosis.