Proteomic analysis of the neutrophil proteins of the tammar wallaby (Macropus eugenii)

A proteomic analysis of neutrophils from the tammar wallaby, Macropus eugenii, has been performed. Neutrophils were isolated from peripheral blood using density gradient centrifugation with Histopaque-1077, followed by treatment with ammonium chloride to lyse residual erythrocytes. Two-dimensional gel electrophoresis (2-DE) of lysed neutrophils was undertaken followed by in-gel trypsin digest and nanoliquid chromatography coupled tandem mass spectrometry (LC-MS) analysis and database searches. Seventy-seven proteins were isolated, 53 of which could be identified with high confidence as primarily of cytosolic origin. Protein identifications were only possible by matching identical peptide sequences within the NCBInr mammalian database with the Mascot search program. Sequence identities were only deemed acceptable if more than three peptides were identified, the precursor/protein ion tolerances were less than ± 0.25 Da and the total Mowse scores were greater than 100. The validity of this approach was tested using a scrambled database where no single identified peptide showed Mowse scores greater than 55. This is the first report of the neutrophil proteins of any marsupial and represents a first step in examining the identity of proteins involved in innate defence in this marsupial.     

Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry

Platelets play a key role in the control of bleeding and wound healing, contributing to the formation of vascular plugs. Under pathologic circumstances, they are involved in thrombotic disorders, including heart disease. Since platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. In this publication we extend the previously reported analysis of the pI 4-5 region of the human platelet proteome to the pI 5-11 region. By using narrow pI range two-dimensional electrophoresis (2-DE) for protein separation followed by high-throughput tandem mass spectrometry (MS/MS) for protein identification, we were able to identify 760 protein features, corresponding to 311 different genes, resulting in the annotation of 54% of the pI 5-11 range 2-DE proteome map. We evaluated the physicochemical properties and functions of the identified platelet proteome. Importantly, the main group of proteins identified is involved in intracellular signalling and regulation of the cytoskeleton. In addition, 11 hypothetical proteins are reported. In conclusion, this study provides a unique inventory of the platelet proteome, contributing to our understanding of platelet function and building the basis for the identification of new drug targets.     

Proteome comparative analysis of gynogenetic haploid and diploid embryos of goldfish (Carassius auratus)

Recently, it was found that in the gynogenetic haploid and diploid embryos of goldfish, which have exactly the same genome, the haploid condition results in obstruction of gene expression and abnormal development while the diploid embryos have normal gene expression and development. A diploid-dependent regulatory apparatus was proposed to regulate gene expression. To study the difference at the protein expression level of the embryos of haploid and diploid in development, we extracted the total proteins of both the gynogenetic haploid and diploid embryos of goldfish in the same eye formation stage. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. The stained gel images were analyzed with the PDQUEST software. A part of protein spots that were differentially expressed in haploid and diploid embryos were identified by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry and database analysis. Sixteen protein spots that were absolutely different (only expressed in diploid embryos but not in haploid embryos or vice versa) and 16 protein spots that were up- and downregulated were identified unambiguously, which include some proteins that are correlative with eyes development, nerve development, developing regulation, cell differentiation, and signal transduction. The different significantly gene expression during embryos developing between diploid and haploid is demonstrated.     

A proteomic analysis of thioacetamide-induced hepatotoxicity and cirrhosis in rat livers

Thioacetamide (TAA) administration is an established technique for generating rat models of liver fibrosis and cirrhosis. Oxidative stress is believed to be involved as TAA-induced liver fibrosis is initiated by thioacetamide S-oxide, which is derived from the biotransformation of TAA by the microsomal flavine-adenine dinucleotide (FAD)-containing monooxygense (FMO) and cytochrome P450 systems. A two-dimensional gel electrophoresis-mass spectrometry approach was applied to analyze the protein profiles of livers of rats administered with sublethal doses of TAA for 3, 6 and 10 weeks respectively. With this approach, 59 protein spots whose expression levels changed significantly upon TAA administration were identified, including three novel proteins. These proteins were then sorted according to their common biochemical properties and functions, so that pathways involved in the pathogenesis of rat liver fibrosis due to TAA-induced toxicity could be elucidated. As a result, it was found that TAA-administration down-regulated the enzymes of the primary metabolic pathways such as fatty acid beta-oxidation, branched chain amino acids and methionine breakdown. This phenomenon is suggestive of the depletion of succinyl-CoA which affects heme and iron metabolism. Up-regulated proteins, on the other hand, are related to oxidative stress and lipid peroxidation. Finally, these proteomics data and the data obtained from the scientific literature were integrated into an "overview model" for TAA-induced liver cirrhosis. This model could now serve as a useful resource for researchers working in the same area.     

The endocrine regulation of milk lipid synthesis and secretion in tammar wallaby (Macropus eugenii)

Lipids in tammar milk are predominantly triacylglycerols, and the fatty acid composition varies during the lactation cycle. Little is known about the regulation of their synthesis. This study investigates the endocrine regulation of lipid synthesis in mammary explants from pregnant tammars. Treatment of mammary explants with insulin resulted in a high level of lipid synthesis, but the lipids accumulated in the cytosol. Culture with prolactin resulted in a small increase in lipid synthesis, but electron microscopy showed lipid globules were synthesized in the mammary epithelial cells and secreted into the lumen. Culture with both insulin and prolactin demonstrated elevated levels of synthesis and secretion of lipid. Analysis of the type of fatty acids synthesized in these mammary explants showed that the initiation of synthesis of C_16:0, which also occurs in the first week of lactation, could be reproduced in the pregnant explants cultured with prolactin alone. However, treatment of mammary explants with hydrocortisone did not show a significant effect on lipid synthesis, secretion or the fatty acid synthesized. These results provide new information identifying the role of insulin and prolactin in regulating milk lipid synthesis and secretion in the tammar.     

Proteomic characterization of the released/secreted proteins of Leishmania (Viannia) braziliensis promastigotes

Extracellular proteins secreted/released by protozoan parasites are key mediators of the host–parasite interaction. To characterise the profile of proteins secreted/released by Leishmania (Viannia) braziliensis promastigotes, a proteomic approach combining two-dimensional electrophoresis (2DE), tandem matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF) mass spectrometry, and data mining was carried out. The 2DE map revealed a set of 270 secreted protein spots from which 42 were confidently identified and classified into 11 categories according to Gene Ontology (GeneDB database) and KEEG Ontology annotation of biological processes. Parasite promastigotes were able to secrete/release proteins involved in immunomodulation, signal transduction, and intracellular survival, such as HSP70, acid phosphatase, activated protein kinase C receptor (LACK), elongation factor 1β, and tryparedoxin peroxidase. Data mining showed that ~ 5% of identified proteins present a classical secretion signal whereas ~ 57% were secreted following non-classical secretion mechanisms, indicating that protein export in this primitive eukaryote might proceed mainly by unconventional pathways. This study reports a suitable approach to identify secreted proteins in the culture supernatant of L. braziliensis and provides new perspectives for the study of molecules potentially involved in the early stages of infection.     

Proteomic analysis of mouse jejunal epithelium and its response to infection with the intestinal nematode, Trichinella spiralis

Infection with the intestinal nematode Trichinella spiralis induces profound, but stereotypic pathological changes to the epithelium, which are common to many nematode infections. This study describes changes in jejunal epithelial protein expression that reflect these stereotypic responses. Adult male BALB/c mice were infected with T. spiralis, and groups (n = 4) examined on day 14/15 (time of worm rejection) were compared with uninfected controls (n = 4). Jejunal epithelium was harvested and extracted for two-dimensional gel electrophoresis. Tryptic peptide mass fingerprinting was used to create a reference map consisting of a total of 52 landmark spots. Of these, 16 were observed to change in intensity during infection. The changes observed at day 14/15 were of relevance to such mechanisms as lipid utilization and transport (increase in triacylglycerol lipase, and reduction in intestinal fatty acid binding protein) and innate immunity (appearance of intelectin-2). As a result, candidate molecules have been identified for further focused studies on their role in the host response to intestinal nematode infection.     

The life history of Labiostrongylus eugenii, a nematode parasite of the Kangaroo Island Wallaby (Macropus eugenii): The parasitic stages

Smales L. R. 1977. The life history of Labiostrongylus eugenii, a nematode parasite of the Kangaroo Island Wallaby (Macropus eugenii): the parasitic stages. International Journal for Parasitology7: 457–461. Labiostrongylus eugenii infective larvae ingested by the Kangaroo Island Wallaby exsheathe on the saccular stomach wall, then invade the mucosa causing one or two chronic irritative hyperpastic nodules, each containing a number of larvae. Six to eight weeks later the larvae leave the stomach wall and moult to fourth-stage. The fourth-stage larva is characterized by eight lips, while the oesophagus and anterior intestine assume the adult form. The posterior ends of late fourth-stage larvae become sexually differentiated. Moulting to adult occurs after 6–8 weeks with the first gravid females being observed 3 months later. The prepatent period appears to last about 6 months. Reasons for failure of experimental infection trials and the epidemiological significance of the life cycle were discussed.     

A proteomic analysis of Helicoverpa armigera adults after exposure to UV light irradiation

Ultraviolet (UV) light (blacklight), which emits UV in the range of 320-400 nm, has been used worldwide in light trapping of insect pests. To gain a better understanding of the response of Helicoverpa armigera adults to UV light irradiation, we carried out a comparative proteomic analysis. Three-day-old adults were exposed to UV light for 1 h. Total proteins were extracted and separated by two-dimensional gel electrophoresis. More than 1200 protein spots were reproducibly detected, including 12 that were more abundant and 21 less abundant. Mass spectrometry analysis and database searching helped us to identify 29 differentially abundant proteins. The identified proteins were categorized into several functional groups including signal transduction, RNA processing, protein processing, stress response, metabolisms, and cytoskeleton structure, etc. This study is the first analysis of differentially expressed proteins in phototactic insects under UV light irradiation conditions and gives new insights into the adaptation mechanisms responsive to UV light irradiation stress.     

A comparative proteomic study of the undeveloped and developed Schistosoma mansoni egg and its contents: The miracidium, hatch fluid and secretions

The schistosome egg is the key agent responsible both for transmission of the parasite from human to molluscan host, and is the primary cause of pathogenesis in schistosomiasis. Characterisation of its proteome is a crucial step in understanding the egg’s interactions with the mammalian host. We devised a scheme to isolate undeveloped eggs from mature schistosome eggs by Percoll gradient and then fractionate the mature egg into miracidial, hatch fluid and secreted protein preparations. The soluble proteins contained within the five preparations were separated by two-dimensional electrophoresis and their spot patterns compared by image analysis. Large numbers of representative spots were then excised and subjected to tandem mass spectrometry to obtain identities. In this way, the principal components of each sub-proteome were established. Chaperones were the most abundant category, with heat shock protein 70 (HSP70) dominant in the undeveloped egg and Schistosoma mansoni protein 40 (Smp-40) in the miracidium. Cytoskeletal proteins were expressed at similar levels in the undeveloped egg and miracidium, with tubulins the most abundant. The proteins of energy metabolism reflected the change from anaerobic to aerobic metabolism as the miracidium developed. None of the above categories was abundant in the hatch fluid but this peri-miracidial compartment was highly enriched for defence proteins such as thioredoxin. Hatch fluid also contained several host proteins and schistosome proteins of unknown function, highlighting its distinct nature and potentially its role. The egg secretions could not be compared with the other preparations due to their unique composition featuring the previously characterised IL-4-inducing principal of S. mansoni eggs (IPSE), Omega-1, egg secreted protein 15 (ESP15), a micro-exon gene 2 (MEG-2) protein and two members of the recently described MEG-3 family. This last preparation contains the subset of egg proteins that probably enables eggs to escape from host tissues and may also initiate granuloma formation, emphasising the need to establish fully the roles of its components in schistosome biology.     

A combination of protein profiling and isotopomer analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometry reveals an active metabolism of the extracellular matrix of 3T3-L1 adipocytes

Differential gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a commonly used protein profiling method. However, observed changes can be explained in multiple ways, one of which is by the protein turnover rate. In order to easily and rapidly obtain information on both the identity and turnover of individual proteins, we applied a combination of protein labeling with L-(ring-2,3,4,5,6 2H5) phenylalanine and MALDI-TOF MS. While the spectrum reveals the identity of a protein, mass isotopomer analysis provides information about the rate of protein labeling as a measure of synthesis or turnover. Using this approach on mature 3T3-L1 adipocytes, we were able to discriminate between rapidly and slowly metabolised proteins. In our isolate, proteins of the cytoskeleton appeared to be slowly metabolised, whereas components of the extracellular matrix, in particular collagen type I alpha 1 (COL1A1) and collagen type I alpha 2 (COL1A2) showed rapid accumulation of newly synthesized proteins. Both proteins appeared to be metabolised in the same ratio as they are present in collagen fibers, i.e. 2:1 (COL1A1: COL1A2). In addition, functionally related proteins were also readily labeled. Taken together, we have shown that a combination of stable isotope labeling and protein profiling by gel electrophoresis and MALDI-TOF analysis can simultaneously provide information on the identity and relative metabolic rate of proteins in eukaryotic cells in a simple, nonhazardous and rapid-throughput way.     

Comparative proteomic analysis between the invasive and commensal strains of Staphylococcus epidermidis

Staphylococcus epidermidis is one of the major causative agents for nosocomial infections. To reveal the pathogenesis factors, we performed the comparative proteomic analysis of invasive ATCC35984 and commensal ATCC12228 strains by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. The differentially expressed proteins were involved in carbohydrate metabolism, sugar binding, lipid degradation and amino acid binding. In addition, we demonstrated that the trap gene was transcribed by 3.657+/-0.156 (P<0.01) -fold higher in ATCC35984 than in ATCC12228. Levels of accumulation-associated protein (AAP) were found to be low by the immuno-dot blotting assay in ATCC12228, which is unable to form biofilm. Our results suggest that the target of RNAIII activating protein and AAP may contribute to S. epidermidis virulence and biofilm formation.     

Electrospray mass spectrometry of trans-[Ru(NO)Cl(dpaH)2] (dpaH=2,2′-dipyridylamine) and ‘caged NO’, [RuCl3(NO)(H2O)2]: loss of HCl and NO from positive ions versus NO and Cl from negative ions

The positive ion electrospray mass spectrometry (ESI-MS) of trans-[Ru(NO)Cl)(dpaH)₂]Cl₂ (dpaH=2,2′-dipyridylamine), obtained from the carrier solvent of H₂O–CH₃OH (50:50), revealed 1+ ions of the formulas [Ru^II(NO⁺)Cl(dpaH)(dpa)]⁺ (m/z=508), [Ru^IIICl(dpaH)(dpa⁻)]⁺ (m/z=478), [Ru^II(NO⁺)(dpa)₂]⁺ (m/z=472), [Ru^III(dpa)₂]⁺ (m/z=442), originating from proton dissociation from the parent [Ru^II(NO⁺)Cl(dpaH)₂]²⁺ ion with subsequent loss of NO (17.4% of dissociative events) or loss of HCl (82.6% of dissociative events). Further loss of NO from the m/z=472 fragment yields the m/z=442 fragment. Thus, ionization of the NH moiety of dpaH is a significant factor in controlling the net ionic charge in the gas phase, and allowing preferential dissociation of HCl in the fragmentation processes. With NaCl added, an ion pair, {Na[Ru^II(NO)Cl(dpa)₂]}⁺ (m/z=530; 532), is detectable. All these positive mass peaks that contain Ru carry a signature ‘handprint’ of adjacent m/z peaks due to the isotopic distribution of ¹⁰⁴Ru, ¹⁰²Ru, ¹⁰¹Ru, ⁹⁹Ru, ⁹⁸Ru and ⁹⁶Ru mass centered around ¹⁰¹Ru for each fragment, and have been matched to the theoretical isotopic distribution for each set of peaks centered on the main isotope peak. When the starting complex is allowed to undergo aquation for two weeks in H₂O, loss of the axial Cl⁻ is shown by the approximately 77% attenuation of the [Ru^II(NO⁺)Cl(dpaH)(dpa)]⁺ ion, being replaced by the [Ru^II(NO⁺)(H₂O)(dpa)₂]⁺ (m/z=490) as the most abundant high-mass species. Loss of H₂O is observed to form [Ru^II(NO⁺)(dpa)₂]⁺ (m/z=472). No positive ion mass spectral peaks were observed for RuCl₃(NO)(H₂O)₂, ‘caged NO’. Negative ions were observed by proton dissociation forming [Ru^II(NO)Cl₃(H₂O)(OH)]⁻ in the ionization chamber, detecting the parent 1− ion at m/z=274, followed by the loss of NO as the main dissociative pathway that produces [Ru^IIICl₃(H₂O)(OH)]⁻ (m/z=244). This species undergoes reductive elimination of a chlorine atom, forming [Ru^IICl₂(H₂O)(OH)]⁻ (m/z=208). The ease of the NO dissociation is increased for the negative ions, which should be more able to stabilize a Ru^III product upon NO loss.     

Differential display of proteins involved in the neural differentiation of mouse embryonic carcinoma P19 cells by comparative proteomic analysis

Mouse embryonic carcinoma P19 cell has been used extensively as a model to study molecular mechanisms of neural differentiation in vitro. After retinoic acid (RA) treatment and aggregation, P19 cells can differentiate into neural cells including neurons and glial cells. In this study, comparative proteomic analysis is utilized to approach the protein profiles associated with the RA-induced neural differentiation of P19 cells. Image analysis of silver stained two-dimensional gels indicated that 28 protein spots had significantly differential expression patterns in both quantity and quality. With mass spectrometry analysis and protein functional exploration, many proteins demonstrated an association with distinct aspects of neural differentiation. These proteins were gag polyprotein, rod cGMP-specific 3',5'-cyclic phosphodiesterase, 53 kDa BRG1-associated factor A, N-myc downstream regulated 1, Vitamin D receptor associated factor 1, stromal cell derived factor receptor 1, phosphoglycerate mutase, Ran-specific GTPase-activating protein, and retinoic acid (RA)-binding protein. While some cytoskeleton-related proteins such as beta cytoskeletal actin, gamma-actin, actin-related protein 1, tropomyosin 1, and cofilin 1 are related to cell migration and aggregation, other proteins have shown a relationship with distinct aspects of neural differentiation including energy production and utilization, protein synthesis and folding, cell signaling transduction, and self-protection. The differential expression patterns of these 28 proteins indicate their different roles during the neural differentiation of P19 cells. As an initial step toward unveiling the regulations involved in the commitment of pluripotent cells to a neural fate, information from this study may be helpful to uncover the molecular mechanisms of neural differentiation.     

花生叶片蛋白组对UV-B辐射增强的响应

为揭示UV-B辐射增强处理降低花生光合速率和花生抵御UV-B辐射增强的分子机制,应用蛋白质双向电泳与质谱联用技术对自然光环境下补增UV-B辐射(54μW/cm2)处理24h的苗期花生叶片差异表达蛋白质变化进行了分析。结果表明:补增UV-B处理下,花生叶片中共检测到丰度变化在2.5倍以上的差异表达蛋白点39个(其中22种蛋白质表达下调,17种表达上调),经过MALDI-TOF-TOF分析及数据库检索,成功鉴定出其中的27种蛋白质。被鉴定的27种蛋白质按其功能大致可归为8类,第Ⅰ类:光合作用相关的蛋白质,包括质体蓝素、1,5-二磷酸核酮糖羧化酶小亚基、放氧复合物增强子蛋白1、PsbP结构域蛋白6和果糖二磷酸醛缩酶;第Ⅱ类:糖代谢相关蛋白质,包括苹果酸脱氢酶;第Ⅲ类:能量合成相关蛋白质,包括ATP合酶;第Ⅳ类:氨基酸代谢相关蛋白质,包括半胱氨酸合成酶;第Ⅴ类:蛋白质加工相关蛋白质,包括热激蛋白;第Ⅵ类:蛋白质翻译相关蛋白质,包括核糖体循环因子;第Ⅶ类:防御相关蛋白质,包括几丁质酶、过氧化物酶、Cu-Zn超氧化物歧化酶、二羟肉桂酸3-O-转甲基酶和类萌发素蛋白;第Ⅷ类,未知功能蛋白质。这些研究结果为进一步研究花生抵御UV-B辐射的分子机理提供了有意义的线索。     

Proteomic analysis of nucleopolyhedrovirus infection resistance in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Silkworm hemolymph is an important defense tissue to resist bacteria and virus infections. To study the response of silkworm hemolymph in the resistance of Bombyx mori L. nucleopolyhedrovirus (BmNPV), we constructed a near-isogenic silkworm line with BmNPV resistance using highly resistant and highly susceptible parental strains. In this paper, two-dimensional gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization (MALDI)-mass spectrometry were employed to investigate the differences of protein patterns in the hemolymph of the highly resistant, highly susceptible and near-isogenic silkworm strains after BmNPV was administrated to the larvae. A comparison between the proteomes of these three silkworm strains led us to identify two differentially expressed proteins, beta-N-acetylglucosaminidase 2 and aminoacylase. The expression levels of these proteins were higher in the BmNPV resistant strains.     

Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells

Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations.     

Proteome analysis of the rat hepatic stellate cells under high concentrations of glucose

To study the change in hepatic stellate cell (HSC) function under diabetic conditions, we cultured rat HSC in the presence of 5 and 30 mM glucose, which correspond to blood glucose concentrations during the early and late stages of diabetes, respectively. The differentially expressed HSC proteins were analyzed using 2-DE and ESI-Q-TOF MS/MS and confirmed with Western blotting. The changed protein expression will provide greater understanding of glycolysis in HSC at the high concentration of glucose.