Anne Stanley

Proteomic profiling of human plasma by iTRAQ reveals down-regulation of ITI-HC3 and VDBP by cigarette smoking.

Biomarkers in noninvasive fluids indicative of cigarette smokes effects are urgently needed. In this pilot study, we utilized the proteomic approach, isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to identify differentially expressed plasma proteins in healthy cigarette smokers compared to healthy nonsmokers; select proteins were further confirmed by immunoblot analysis. Significant, differentially expressed proteins identified in the plasma separated subjects based on their condition as smokers or nonsmokers. Several of the proteins identified in this study are associated with immunity and inflammatory responses and have been shown to be associated with tobacco-related diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. Proteins up-regulated in smokers included complement component 8 polypeptide chains α, β, and γ, and mannose-binding protein C, and proteins down-regulated included inter-α-trypsin inhibitor heavy chain H3 (ITI-HC3) and vitamin D-binding protein (VDBP). In addition, gelsolin and vitronectin, known tissue leakage proteins, were up- and down-regulated, respectively. Our results demonstrate for the first time that chronic cigarette smoking can influence the expression profile of the human plasma proteome. Proteins identified in this pilot study may serve as candidate biomarkers of diseases resulting from exposure to cigarette smoke in future molecular epidemiological studies.

Immunodepletion Plasma Proteomics by TripleTOF 5600 and Orbitrap Elite/LTQ-Orbitrap Velos/Q Exactive Mass Spectrometers

Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.

Fall Armyworm-Associated Gut Bacteria Modulate Plant Defense Responses

Mechanical damage caused by insect feeding along with components present in insect saliva and oral secretions are known to induce jasmonic acid-mediated defense responses in plants. This study investigated the effects of bacteria from oral secretions of the fall armyworm Spodoptera frugiperda on herbivore-induced defenses in tomato and maize plants. Using culture-dependent methods, we identified seven different bacterial isolates belonging to the family Enterobacteriacea from the oral secretions of field-collected caterpillars. Two isolates, Pantoea ananatis and Enterobacteriaceae-1, downregulated the activity of the plant defensive proteins polyphenol oxidase and trypsin proteinase inhibitors (trypsin PI) but upregulated peroxidase (POX) activity in tomato. A Raoultella sp. and a Klebsiella sp. downregulated POX but upregulated trypsin PI in this plant species. Conversely, all of these bacterial isolates upregulated the expression of the herbivore-induced maize proteinase inhibitor (mpi) gene in maize. Plant treatment with P. ananatis and Enterobacteriaceae-1 enhanced caterpillar growth on tomato but diminished their growth on maize plants. Our results highlight the importance of herbivore-associated microbes and their ability to mediate insect plant interactions differently in host plants fed on by the same herbivore.

Quantitative proteomic analysis of the fall armyworm saliva

Lepidopteran larvae secrete saliva on plant tissues during feeding. Components in the saliva may aid in food digestion, whereas other components are recognized by plants as cues to elicit defense responses. Despite the ecological and economical importance of these plant-feeding insects, knowledge of their saliva composition is limited to a few species. In this study, we identified the salivary proteins of larvae of the fall armyworm (FAW), Spodoptera frugiperda; determined qualitative and quantitative differences in the salivary proteome of the two host races-corn and rice strains-of this insect; and identified changes in total protein concentration and relative protein abundance in the saliva of FAW larvae associated with different host plants. Quantitative proteomic analyses were performed using labeling with isobaric tags for relative and absolute quantification followed by liquid chromatography-tandem mass spectrometry. In total, 98 proteins were identified (>99% confidence) in the FAW saliva. These proteins were further categorized into five functional groups: proteins potentially involved in (1) plant defense regulation, (2) herbivore offense, (3) insect immunity, (4) detoxification, (5) digestion, and (6) other functions. Moreover, there were differences in the salivary proteome between the FAW strains that were identified by label-free proteomic analyses. Thirteen differentially identified proteins were present in each strain. There were also differences in the relative abundance of eleven salivary proteins between the two FAW host strains as well as differences within each strain associated with different diets. The total salivary protein concentration was also different for the two strains reared on different host plants. Based on these results, we conclude that the FAW saliva contains a complex mixture of proteins involved in different functions that are specific for each strain and its composition can change plastically in response to diet type.

Changes in proteomic profiles in different prostate lobes of male rats throughout growth and development and aging stages of the life span

Aging‐related changes in important cellular pathways in the prostate may promote a permissive environment for an increased risk for prostatic disease development such as prostate cancer. Our objectives were to examine for such changes, by systematically determining the effects of growth and development and aging on proteomic profiles in different lobes of the rat prostate.

Proteomic Changes Induced by Effective Chemopreventive Ratios of n-3:n-6 Fatty Acids and Tamoxifen against MNU-Induced Mammary Cancer in the Rat

We used a proteomic approach to gain insights into the mechanisms of protection at the protein level by a high n-3:n-6 ratio in the absence and presence of Tamoxifen. Four groups were treated with 1-methyl-1-nitrosourea (MNU) and fed the following diets with varied n-3:n-6 ratios; group 1 = 1:1; groups 2 and 3 = 10:1 and 25:1, respectively; group 4: (25:1) plus Tamoxifen (1 mg/kg diet). The plasma from six rats/group was pooled and analyzed with the isobaric tags for relative and absolute quantitation method; 148 proteins were identified with 95% confidence by ProteinPilot 4.0. In plasma of rats fed 10:1, 25:1 n-3:n-6, and 25:1 plus Tamoxifen, the number of proteins that met our criteria (P ≤ 0.05, error factor ≤ 2) were 10, 14, and 19 proteins, respectively. Selected proteins were further validated by Western blotting. Compared to 1:1, both 10:1 and 25:1 diets upregulated vitamin D binding protein, gelsolin, and 14-3-3 sigma, reported to have tumor suppressive effects, whereas alpha-1B-glycoprotein, which has been reported to be elevated in the serum of breast cancer patients was decreased. Compared to 25:1, the 25:1 plus Tamoxifen diet downregulated apolipoprotein E, haptoglobin, and inter-α-inhibitor H4 heavy chain. Ingenuity pathway analysis determined that the trends of specific proteins were related to lipid metabolism in the 25:1 n-3:n-6 group, whereas the 25:1 n-3:n-6 plus Tamoxifen group included proteins involved in cancer and inflammation. Our results show that several proteins were altered in a manner consistent with chemoprevention. Such proteins may serve as biomarkers to monitor efficacy of n-3 and Tamoxifen in future clinical chemoprevention trials. Cancer Prev Res; 6(9); 979–88. ©2013 AACR.

Proteomic analysis of labial saliva of the generalist cabbage looper (Trichoplusia ni) and its role in interactions with host plants

Insect saliva is one of the first secretions to come in contact with plants during feeding. The composition and role of caterpillar saliva has not been as thoroughly studied as that of sucking insects. This study focuses on characterizing the proteome of the cabbage looper (Trichoplusia ni) saliva using iTRAQ labeling and LC-MS/MS. We also measured how the saliva proteome changed when larvae were reared on different diets - cabbage, tomato, and an artificial pinto bean diet. We identified 254 proteins in the saliva out of which 63 were differentially expressed. A large percentage (56%) of the proteins identified function in protein metabolism, followed by proteins involved in vesicle transport (6%) and oxidoreductase activity (5%), among other categories. Several proteins identified are antioxidants or reactive oxygen species (ROS) scavengers. Among these ROS scavengers, we identified a catalase and further analyzed its gene expression and enzymatic activity. We also applied commercial, purified catalase on tomato and measured the activity of defensive proteins - trypsin proteinase inhibitor, polyphenol oxidase and peroxidase. Catalase gene expression was significantly higher in the salivary glands of larvae fed on tomato. Also, catalase suppressed the induction of tomato trypsin proteinase inhibitor levels, but not the induction of polyphenol oxidase or peroxidase. These results add to our understanding of proteomic plasticity in saliva and its role in herbivore offense against plant defenses.

Abstract 4572: Comparison of proteomic profiles in human plasma of smokers and nonsmokers using the iTRAQ approach

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Lung cancer is a potentially preventable disease because it is strongly related to tobacco smoke. Although there have been great strides taken to inform the population of the severity of the risks associated with smoking, there are currently 43.4 million Americans that smoke and 1.3 billion smokers worldwide. Therefore, it is important to develop biomarkers that can identify individual smokers and ex-smokers who are at especially high risk for lung cancer. Relative changes that take place in the human plasma proteome as a result of smoking may be playing an important role in the etiology of lung cancer. Thus, in this study we used a proteomic approach utilizing isobaric Tag for Relative and Absolute Quantitation (iTRAQ) to compare the plasma protein expression levels between healthy smokers and non-smokers. With this multi-plex non-gel based technique, up to 8 samples can be simultaneously labeled with isobaric tagging reagents, 113-119 and 121, for quantitation and identification by mass spectrometry. Plasma samples from seven white male smokers and seven white male non-smokers were distributed into two 8-plex iTRAQ sets. The 113 label in each set was used to label a pool consisting of each sample in the study in order to compare between the two 8-plex sets. Plasma from each subject was depleted of the 14 most abundant plasma proteins using an immunoaffinity column to search for lower abundant proteins. Based on our results, we identified 18 significant (p<0.05) differentially expressed proteins between smokers and non-smokers. The proteins that were down-regulated in smokers include sex hormone-binding globulin (SHBG), inter-α (globulin) inhibitor H3 (HC3), and vitronectin. The proteins that were up-regulated in smokers include the complement component 8, α, β and γ polypeptides, gelsolin, and mannose-binding protein C (MBP-C). Among those identified, SHBG, HC3, and gelsolin protein have been previously identified in smokers and have been suggested to be candidate markers for lung cancer. The results of this study show that cigarette smoking can influence the relative expression profile of plasma proteins. However, the relationship of these proteins to the development of lung cancer needs to be determined so that they can be further developed as biomarkers of disease risk. Support: NCI PO1 70972 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4572.

Abstract 4589: Changes in proteomic profiles with age in male rats using the iTRAQ approach

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Aging is an important risk factor for many diseases including prostate cancer (PC). Enhanced risk for disease may, in part, be due to aging-related changes in the proteome. Thus, a better knowledge of the changes in protein levels occurring with age would help in the early detection of the disease, as well as in monitoring chemopreventive intervention. In the present study we used male F344 rats, a commonly used aging and PC model, and compared protein levels in the blood plasma of young (4 months), mature (12 months), old (18 months) and very old (24 months) rats by a proteomic approach known as isobaric Tag for Relative and Absolute Quantitation (iTRAQ). In this multi-plex non-gel based technique samples can be simultaneously labeled with isobaric tagging reagents for quantitation and identification by mass spectrometry. Plasma samples were pooled from three animals and 50 μg of protein from each group was labeled for a 4-plex iTRAQ analysis. A total of seventy nine proteins were identified, thirty one with >99% confidence. Among those, the levels of thirteen proteins were changed significantly (p 99% confidence including serotransferrin, hemopexin, fetuin-A, fetuin-B and α-1-acid glycoprotein. Both age-specific and growth-development related changes were observed throughout the life span with maximum changes (either increases or decreases) being observed at 18 months. The results of this study show that age indeed can influence the expression profile of plasma proteins. The relationship of these proteins to PC is currently being investigated by analyzing their levels in different lobes of the prostate gland. Supported in part by R01 CA 127729-01 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4589.