Comparative proteomic analysis of Helicoverpa armigera cells undergoing apoptosis

Apoptosis is of crucial importance in the life of multicellular organisms. In holometabolous insects, particularly in Lepidoptera, apoptosis is essential in biological processes such as metamorphosis and defense against pathogens. Apoptosis is tightly regulated and involves many proteins, among them caspases, which play a central role. In mammals, almost 300 targets of caspases have been described, and the expression of more than a hundred proteins has been shown to be altered in apoptotic cells. To date, the molecular pathways controlling apoptosis are poorly understood in Lepidoptera. Here, we used a comparative approach aiming to identify candidate proteins potentially implicated in these pathways. We examined changes occurring, in the proteome of a Helicoverpa armigera-derived cell line, upon induction by actinomycin D. We identified 13 proteins for which the relative abundance was significantly altered. Among these, the abundance of procaspase-1 decreased in apoptotic cells, reflecting its processing into the active form. We characterized its properties by heterologous expression and correlated the observed substrate specificity with changes in caspase activity in HaAM1 cells after induction. We also identified three chaperones as well as several putative pro- and anti-apoptotic proteins. Altogether, these data suggest that apoptotic pathways in Lepidoptera share similarities with the ones described in mammals.     

Differential proteomic analysis of proteins in wheat spikes induced by Fusarium graminearum

Scab, caused by Fusarium graminearum, is a serious spike disease in wheat. To identify proteins in resistant wheat cultivar Wangshuibai induced by F. graminearum infection, proteins extracted from spikes 6, 12 and 24 h after inoculation were separated by 2-DE. Thirty protein spots showing 3-fold change in abundance when compared with treatment without inoculation were characterized by MALDI-TOF MS and matched to proteins by querying the mass spectra in protein databases or the Triticeae EST translation database. Based on their volume profiles, these proteins were classified into four categories. The first one fell off rapidly at the initial inoculation and then rose at 12 or 24 hai, the second one decreased considerably after inoculation and remained at low level, the third one rose at the initial inoculation and then declined at 12 or 24 hai, the forth one showed steady increase after inoculation and maintained at a high level. Many of the proteins identified in the first two categories are related to carbon metabolism and photosynthesis. While most of proteins identified in the last two categories are related to stress defense of plants, indicating that proteins associated with the defense reactions were activated or translated shortly after inoculation.     

Differential proteomic analysis of a polymicrobial biofilm

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia exist in a polymicrobial biofilm associated with chronic periodontitis. The aim of this study was to culture these three species as a polymicrobial biofilm and to determine proteins important for bacterial interactions. In a flow cell all three species attached and grew as a biofilm; however, after 90 h of culture P. gingivalis and T. denticola were closely associated and dominated the polymicrobial biofilm. For comparison, planktonic cultures of P. gingivalis and T. denticola were grown separately in continuous culture. Whole cell lysates were subjected to SDS-PAGE, followed by in-gel proteolytic H(2)(16)O/H(2)(18)O labeling. From two replicates, 135 and 174 P. gingivalis proteins and 134 and 194 T. denticola proteins were quantified by LC-MALDI TOF/TOF MS. The results suggest a change of strategy in iron acquisition by P. gingivalis due to large increases in the abundance of HusA and HusB in the polymicrobial biofilm while HmuY and other iron/haem transport systems decreased. Significant changes in the abundance of peptidases and enzymes involved in glutamate and glycine catabolism suggest syntrophy. These data indicate an intimate association between P. gingivalis and T. denticola in a biofilm that may play a role in disease pathogenesis.     

Microglial activation induced by neurodegeneration: a proteomic analysis

Neuroinflammation mediated by microglial activation appears to play an essential role in the pathogenesis of Parkinson disease; however, the mechanisms by which microglia are activated are not fully understood. Thus, we first evaluated the effects of two parkinsonian toxicants, manganese ethylene bisdithiocarbamate (Mn-EBDC) and 1-methyl-4-phenylpyridine (MPP+), on microglial activation as well as associated dopaminergic (DAergic) neurotoxicity in primary cell culture systems. The results demonstrated that, when rat primary mesencephalic neuron-enriched or neuron-microglia mixed cultures were treated with Mn-EBDC at 2-8 microm or MPP+ at 0.25-5 microm, respectively, for 7 days, both toxicants were capable of inducing DAergic neurodegeneration as well as activating microglia via a mechanism secondary to DAergic neurodegeneration. Furthermore activated microglia subsequently enhanced DAergic neurotoxicity induced by Mn-EBDC or MPP+. Detailed scrutiny of neuron-microglia interactions identified a fraction of the conditioned media derived from a DAergic cell line treated with Mn-EBDC or MPP+ that potently activated microglia. To further define potential mediators leading to microglial activation secondary to neurodegeneration, we utilized a quantitative proteomic technique termed SILAC (for stable isotope labeling by amino acids in cell culture) to compare the protein profiles of MPP+-treated cellular fraction that mediated microglial activation as compared with controls. The search revealed numerous novel proteins that are potentially important in neurodegeneration-mediated microglial activation, a process believed to be critical in Parkinson disease progression.     

Combined gene expression and proteomic analysis of EGF induced apoptosis in A431 cells suggests multiple pathways trigger apoptosis

A431 cells, derived from epidermoid carcinoma, overexpress the epidermal growth factor receptor (EGFR) and when treated with a high dose of EGF will undergo apoptosis. We exploited microarray and proteomics techniques and network prediction to study the regulatory mechanisms of EGF-induced apoptosis in A431 cells. We observed significant changes in gene expression in 162 genes, approximately evenly split between pro-apoptotic and anti-apoptotic genes and identified 30 proteins from the proteomic data that had either pro or anti-apoptotic annotation. Our correlation analysis of gene expression and proteome modeled a number of distinct sub-networks that are associated with the onset of apoptosis, allowing us to identify specific pathways and components. These include components of the interferon signalling pathway, and down stream components, including cytokines and suppressors of cytokine signalling. A central component of almost all gene expression sub-networks identified was TP53, which is mutated in A431 cells, and was down regulated. This down regulation of TP53 appeared to be correlated with proteomic sub-networks of cytoskeletal or cell adhesion components that might induce apoptosis by triggering cytochrome C release. Of the only three genes also differentially expressed as proteins, only serpinb1 had a known association with apoptosis. We confirmed that up regulation and cleavage of serpinb1 into L-DNAaseII was correlated with the induction of apoptosis. It is unlikely that a single pathway, but more likely a combination of pathways is needed to trigger EGF induced apoptosis in A431cells.     

Differential proteomic analysis of mammalian tissues using SILAM

Differential expression of proteins between tissues underlies organ-specific functions. Under certain pathological conditions, this may also lead to tissue vulnerability. Furthermore, post-translational modifications exist between different cell types and pathological conditions. We employed SILAM (Stable Isotope Labeling in Mammals) combined with mass spectrometry to quantify the proteome between mammalian tissues. Using (15)N labeled rat tissue, we quantified 3742 phosphorylated peptides in nuclear extracts from liver and brain tissue. Analysis of the phosphorylation sites revealed tissue specific kinase motifs. Although these tissues are quite different in their composition and function, more than 500 protein identifications were common to both tissues. Specifically, we identified an up-regulation in the brain of the phosphoprotein, ZFHX1B, in which a genetic deletion causes the neurological disorder Mowat-Wilson syndrome. Finally, pathway analysis revealed distinct nuclear pathways enriched in each tissue. Our findings provide a valuable resource as a starting point for further understanding of tissue specific gene regulation and demonstrate SILAM as a useful strategy for the differential proteomic analysis of mammalian tissues.     

Comprehensive proteomic analysis of human bile

Bile serves diverse functions from metabolism to transport. In addition to acids and salts, bile is composed of proteins secreted or shed by the hepatobiliary system. Although there have been previous efforts to catalog biliary proteins, an in-depth analysis of the bile proteome has not yet been reported. We carried out fractionation of non-cancerous bile samples using a multipronged approach (SDS-PAGE, SCX and OFFGEL) followed by MS analysis on an LTQ-Orbitrap Velos mass spectrometer using high resolution at both MS and MS/MS levels. We identified 2552 proteins - the largest number of proteins reported in human bile till date. To our knowledge, there are no previous studies employing high-resolution MS reporting a more detailed catalog of any body fluid proteome in a single study. We propose that extensive fractionation coupled to high-resolution MS can be used as a standard methodology for in-depth characterization of any body fluid. This catalog should serve as a baseline for the future studies aimed at discovering biomarkers from bile in gallbladder, hepatic, and biliary cancers.     

A proteomic analysis of human bile

We have carried out a comprehensive characterization of human bile to define the bile proteome. Our approach involved fractionation of bile by one-dimensional gel electrophoresis and lectin affinity chromatography followed by liquid chromatography tandem mass spectrometry. Overall, we identified 87 unique proteins, including several novel proteins as well as known proteins whose functions are unknown. A large majority of the identified proteins have not been previously described in bile. Using lectin affinity chromatography and enzymatically labeling of asparagine residues carrying glycan moieties by (18)O, we have identified a total of 33 glycosylation sites. The strategy described in this study should be generally applicable for a detailed proteomic analysis of most body fluids. In combination with "tagging" approaches for differential proteomics, our method could be used for identification of cancer biomarkers from any body fluid.     

Differential proteomic analysis of cadmium-responsive proteins in wheat leaves

To gain a comprehensive understanding of plant response to Cd, physiological and proteomic changes in wheat (Triticum aestivum L.) leaves exposed to a range of Cd concentrations (10, 100 and 200 μM) were investigated. Leaf elongation was decreased, whereas the H₂O₂ and malondialdehyde content increased significantly at higher Cd concentrations. Changes in protein profiles were analyzed by two-dimensional electrophoresis. Twenty-one proteins which showed 1.5-fold change in protein abundance in response to Cd were identified. These proteins can be functionally grouped into three groups: 1) oxidative stress response, 2) photosynthesis and sugar metabolism and 3) protein metabolism and others. These results provide a new insight into our understanding of the molecular basis of heavy metal response in plants.     

Differential proteomic analysis of adrenal gland during postnatal development

The adrenal glands (AGs) are endocrine organs essential for life. They undergo a fetal to adult developmental maturation process, occurring in rats during the first postnatal month. The molecular modifications underlying these ontogenic changes are essentially unknown. Here we report the results of a comparative proteomic analysis performed on neonatal (Postnatal day 3) versus adult (Postnatal day 30) AGs, searching for proteins with a relative higher abundance at each age. We have identified a subset of proteins with relevant expression in each developmental period using 2‐DE and DIGE analysis. The identified proteins belong to several functional categories, including proliferation/differentiation, cell metabolism, and steroid biosynthesis. To study if the changes in the proteome are correlated with changes at the mRNA level, we have randomly selected several proteins with differential expression and measured their relative mRNA levels using quantitative RT‐PCR. Cell‐cycle regulating proteins (retinoblastoma binding protein 9 and prohibitin) with contrasting effects on proliferation are expressed differentially in neonatal and adult AG. Progesterone metabolizing enzymes, up‐regulated in the neonatal gland, might contribute to the hyporesponsiveness of the adrenal cortex characteristic of this developmental period. We have also observed in the adult gland a marked up‐regulation of enzymes involved in NAD(P)H production, thus providing the reducing power necessary for steroid hormone biosynthesis.     

Differential proteomic analysis of HeLa cells treated with Honokiol using a quantitative proteomic strategy

Honokiol (HNK) is an active component purified from Magnolia officinalis. HNK exhibits antitumor effects by inducing apoptosis and inhibiting the growth of many cancer cell lines, while proteins involved in antitumor effects in proteomic level are still unclear. In our study, HNK could inhibit HeLa cell proliferation and induce apoptosis in a concentration- and time-dependent manner. We utilized a quantitative proteomic technique termed SILAC (Stable isotope labeling with amino acids in cell culture)-MS (mass spectrometry) to study the differential proteomic profiling of HeLa cells treated by HNK. A total of 85 proteins were changed after HeLa cells were treated with 12 microg/ml HNK for 8 h, and 8 proteins showed up-regulation while 77 proteins down-regulated. The changed proteins were classified into 9 different categories, which covered a broad variety of cellular functions. In conclusion, HNK performs cytotoxicity to HeLa cells through co-operating of many proteins and different pathways.     

Apoptosis induced by 1,3,6,7-tetrahydroxyxanthone in Hepatocellular carcinoma and proteomic analysis

Gamboge is a traditional Chinese medicine and our previous study showed that gambogic acid and gambogenic acid suppress the proliferation of HCC cells. In the present study, another active component, 1,3,6,7-tetrahydroxyxanthone (TTA), was identified to effectively suppress HCC cell growth. In addition, our Hoechst-PI staining and flow cytometry analyses indicated that TTA induced apoptosis in HCC cells. In order to identify the targets of TTA in HCC cells, a two-dimensional gel electrophoresis was performed, and proteins in different expressions were identified by MALDA-TOF MS and MS/MS analyses. In summary, eighteen proteins with different expressions were identified in which twelve were up-regulated and six were down-regulated. Among them, the four most distinctively expressed proteins were further studied and validated by western blotting. The β-tubulin and translationally controlled tumor protein were decreased while the 14-3-3σ and P16 protein expressions were up-regulated. In addition, TTA suppressed tumorigenesis partially through P16-pRb signaling. 14-3-3σ silence reversed the suppressive effect of cell growth and apoptosis induced by introducing TTA. In conclusion, TTA effectively suppressed cell growth through, at least partially, up-regulation of P16 and 14-3-3σ.     

Targeted tissue proteomic analysis of human astrocytomas

Complicating proteomic analysis of whole tissues is the obvious problem of cell heterogeneity in tissues, which often results in misleading or confusing molecular findings. Thus, the coupling of tissue microdissection for tumor cell enrichment with capillary isotachophoresis-based selective analyte concentration not only serves as a synergistic strategy to characterize low abundance proteins, but it can also be employed to conduct comparative proteomic studies of human astrocytomas. A set of fresh frozen brain biopsies were selectively microdissected to provide an enriched, high quality, and reproducible sample of tumor cells. Despite sharing many common proteins, there are significant differences in the protein expression level among different grades of astrocytomas. A large number of proteins, such as plasma membrane proteins EGFR and Erbb2, are up-regulated in glioblastoma. Besides facilitating the prioritization of follow-on biomarker selection and validation, comparative proteomics involving measurements in changes of pathways are expected to reveal the molecular relationships among different pathological grades of gliomas and potential molecular mechanisms that drive gliomagenesis.     

Comprehensive proteomic analysis of human pancreatic juice

Proteomic technologies provide an excellent means for analysis of body fluids for cataloging protein constituents and identifying biomarkers for early detection of cancers. The biomarkers currently available for pancreatic cancer, such as CA19-9, lack adequate sensitivity and specificity contributing to late diagnosis of this deadly disease. In this study, we carried out a comprehensive characterization of the "pancreatic juice proteome" in patients with pancreatic adenocarcinoma. Pancreatic juice was first fractionated by 1-dimensional gel electrophoresis and subsequently analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). A total of 170 unique proteins were identified including known pancreatic cancer tumor markers (e.g., CEA, MUC1) and proteins overexpressed in pancreatic cancers (e.g., hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) and lipocalin 2). In addition, we identified a number of proteins that have not been previously described in pancreatic juice (e.g., tumor rejection antigen (pg96) and azurocidin). Interestingly, a novel protein that is 85% identical to HIP/PAP was identified, which we have designated as PAP-2. The proteins identified in this study could be directly assessed for their potential as biomarkers for pancreatic cancer by quantitative proteomics methods or immunoassays.     

Cytofluorometric analysis of cell proliferation and differentiation of the human erythroblasts

Summary The method of cytofluorometric measurement of the contents of Hb and nuclear DNA on a single erythroid cell (Fukuda et al., 1975, 1977 a) was used for the quantitative analysis of the erythropoiesis in normal human bone marrow.The intracellular Hb in an erythroid cell was converted to fluorescent porphyrin after removing the Giemsa staining by irradiation with violet light in the presence of SH-donor (mercaptoethylamine hydrochloride, MEA) and its nuclear DNA was subsequently stained with pararosaniline Feulgen staining. With the two quantitative parameters, Hb content and DNA amount, the erythroid cells in normal human bone narrow were classified into 6 classes of different maturation stages (EI-EIV).The morphological characteristics of the most primitive erythroblast (EI cells) were described. The proerythroblasts which were identified on the bases of morphological criteria had in general aneuploid amounts of nuclear DNA with disproportional contents of Hb, thereby indicating that they are rather aberrations from the normal steps of cell maturation. The DNA amounts of orthochromatic erythroblasts (EV cells) showed continuous decrease from diploid range to almost zero suggesting that the removal of nuclear DNA from the erythroblast is not exclusively due to mechanical expulsion of a whole intact nucleus.Partly supported by Alexander von Humboldt-Stiftung and Deutsche Forschungsgemeinschaft DFG-Grant Bo 395/3     

Comprehensive proteomic analysis of human cervical-vaginal fluid

Cervical-vaginal fluid (CVF) is a potential rich source of biomarkers for enhancing our understanding of human parturition and pathologic conditions affecting pregnancy. In this study, we performed a comprehensive survey of the CVF proteome in pregnancy utilizing multidimensional liquid chromatography (2D-LC) coupled with mass spectrometry and gel-electrophoresis-based protein separation and identification. In total, 150 unique proteins were identified using multiple protein identification algorithms. Metabolism (32%) and immune response-related (22%) proteins are the major functional categories represented in the CVF proteome. A comparison of the CVF, serum, and amniotic fluid proteomes showed that 77 proteins are unique to CVF, while 56 and 17 CVF proteins also occur in serum and amniotic fluid, respectively. This data set provides a foundation for evaluation of these proteins as potential CVF biomarkers for noninvasive diagnosis of pregnancy-related disorders.