iTRAQ-based mass spectrometry screen to identify serum biomarkers in systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disorder with no reliable serum biomarkers currently available other than autoantibodies. METHODS: In the present study, isobaric tags for relative and absolute quantitation-based mass spectrometry was used to screen the sera of patients with SLE to uncover potential disease biomarkers. RESULTS: 85 common proteins were identified, with 16 being elevated (≥1.3) and 23 being decreased (≤0.7) in SLE. Of the 16 elevated proteins, serum alpha-1-microglobulin/bikunin precursor (AMBP), zinc alpha-2 glycoprotein (AZGP) and retinol-binding protein 4 (RBP4) were validated in independent cross-sectional cohorts (Cohort I, N=52; Cohort II, N=117) using an orthogonal platform, ELISA. Serum AMBP, AZGP and RBP4 were validated to be significantly elevated in both patients with inactive SLE and patients with active SLE compared with healthy controls (HCs) (p<0.05, fold change >2.5) in Cohort I. All three proteins exhibited good discriminatory power for distinguishing active SLE and inactive SLE (area under the curve=0.82-0.96), from HCs. Serum AMBP exhibited the largest fold change in active SLE (5.96) compared with HCs and correlated with renal disease activity. The elevation in serum AMBP was validated in a second cohort of patients with SLE of different ethnic origins, correlating with serum creatinine (r=0.60, p<0.001). CONCLUSION: Since serum AMBP is validated to be elevated in SLE and correlated with renal disease, the clinical utility of this novel biomarker warrants further analysis in longitudinal cohorts of patients with lupus and lupus nephritis.

Trajectory of Depression among Prostate Cancer Patients: A Secondary Analysis of a Randomized Controlled Trial.

Background: While psychological difficulties, such as depression, among prostate cancer patients are known, their longitudinal burden remains understudied. We assessed the burden of depression across low-, intermediate- and high-risk prostate cancer groups, and the association between regret and long-term depression. Methods: Secondary analysis of data from a multi-centered randomized controlled study among localized prostate cancer patients was carried out. Assessments were performed at baseline, and at 3-, 6-, 12- and 24-month follow-up. Depression was assessed using the Center for Epidemiologic Studies Depression (CES-D) scale. A CES-D score ≥ 16 indicates high depression. Regret was measured using the regret scale of the Memorial Anxiety Scale for Prostate Cancer (MAX-PC). The proportion of patients with high depression was compared over time, for each risk category. Logistic regression was used to assess the association between regret, and long-term depression after adjusting for age, race, insurance, smoking status, marital status, income, education, employment, treatment, number of people in the household and study site. Results: The study had 743 localized prostate cancer patients. Median depression scores at 6, 12 and 24 months were significantly larger than the baseline median score, overall and for the three prostate cancer risk groups. The proportion of participants with high depression increased over time for all risk groups. Higher regret at 24-month follow-up was significantly associated with high depression at 24-month follow-up, after adjusting for covariates. Conclusions: A substantial proportion of localized prostate cancer patients continued to experience long-term depression. Patient-centered survivorship care strategies can help reduce depression and regret, and improve outcomes in prostate cancer care.

Continuity of care and advanced prostate cancer.

BACKGROUND: Continuity of care is an important element of advanced prostate cancer care due to the availability of multiple treatment options, and associated toxicity. However, the association between continuity of care and outcomes across different racial groups remains unclear. OBJECTIVE: To assess the association of provider continuity of care with outcomes among Medicare fee-for-service beneficiaries with advanced prostate cancer and its variation by race. DESIGN: Retrospective cohort study using Surveillance, Epidemiology, and End Results (SEER)-Medicare data. SUBJECTS: African American and white Medicare beneficiaries aged 66 or older, and diagnosed with advanced prostate cancer between 2000 and 2011. At least 5 years of follow-up data for the cohort was used. MEASURES: Short-term outcomes were emergency room (ER) visits, hospitalizations, and cost during acute survivorship phase (2-year post-diagnosis), and mortality (all-cause and prostate cancer-specific) during the follow-up period. We calculated continuity of care using Continuity of Care Index (COCI) and Usual Provider Care Index (UPCI), for all visits, oncology visits, and primary care visits in acute survivorship phase. We used Poisson models for ER visits and hospitalizations, and log-link GLM for cost. Cox model and Fine-Gray competing risk models were used for survival analysis, weighted by propensity score. We performed similar analysis for continuity of care in the 2-year period following acute survivorship phase. RESULTS: One unit increase in COCI was associated with reduction in short-term ER visits (incidence rate ratio [IRR] = 0.65, 95% confidence interval [CI] 0.64, 0.67), hospitalizations (IRR = 0.65, 95% CI 0.64, 0.67), and cost (0.64, 95% CI 0.61, 0.66) and lower hazard of long-term mortality. Magnitude of these associations differed between African American and white patients. We observed comparable results for continuity of care in the follow-up period. CONCLUSIONS: Continuity of care was associated with improved outcomes. The benefits of higher continuity of care were greater for African Americans, compared to white patients. Advanced prostate cancer survivorship care must integrate appropriate strategies to promote continuity of care.

Loss of 4E-BPs prevents the hindlimb immobilization-induced decrease in protein synthesis in skeletal muscle.

The present study was designed to test the hypothesis that upregulating protein synthesis attenuates the loss of muscle mass in a model of disuse atrophy. The studies compared the effect of unilateral hindlimb immobilization in wild-type (WT) mice and double-knockout (DKO) mice lacking the translational regulators 4E-BP1 and 4E-BP2. Immobilization-induced downregulation of protein synthesis occurred in both groups of mice, but protein synthesis was higher in gastrocnemius muscle from the immobilized hindlimb of fasted DKO compared with WT mice. Surprisingly, although protein synthesis was partially elevated in DKO compared with WT mice, atrophy occurred to the same extent in both groups of animals. This may be partially due to impaired leucine-induced stimulation of protein synthesis in DKO compared with WT mice due to downregulated eukaryotic initiation factor eIF4E expression in muscle of DKO compared with WT mice. Expression of the E3 ubiquitin ligases MAFbx and MuRF-1 mRNAs and total protein ubiquitylation was upregulated in the immobilized compared with the nonimmobilized hindlimb of both WT and DKO mice, with little difference in the magnitude of the upregulation between genotypes. Analysis of newly synthesized proteins revealed downregulation of several glycolytic enzymes in the gastrocnemius of DKO mice compared with WT mice, as well as in the immobilized compared with the nonimmobilized hindlimb. Overall, the results suggest that the elevated rate of protein synthesis during hindlimb immobilization in fasted DKO mice is insufficient to prevent disuse-induced muscle atrophy, probably due to induction of compensatory mechanisms including downregulation of eIF4E expression.NEW & NOTEWORTHY Basal rates of protein synthesis are elevated in skeletal muscle in the immobilized leg of mice lacking the translational repressors, 4E-BP1 and 4E-BP2 (knockout mice), compared with wild-type mice. However, disuse-induced muscle atrophy occurs to the same extent in both wild-type and knockout mice suggesting that compensatory mechanisms are induced that overcome the upregulation of muscle protein synthesis. Proteomic analysis revealed that mRNAs encoding several glycolytic enzymes are differentially translated in wild-type and knockout mice.

Enrichment of Newly Synthesized Proteins following treatment of C2C12 Myotubes with Endotoxin and Interferon-γ.

Inflammation in muscle induces the synthesis of mediators that can impair protein synthesis and enhance proteolysis, and when sustained lead to muscle atrophy. Furthermore, muscle-derived mediators that are secreted may participate in disrupting the function of other peripheral organs. Selective identification of newly synthesized proteins can provide insight on biological processes that depend on the continued synthesis of specific proteins to maintain homeostasis as well as those proteins that are up- or down-regulated in response to inflammation. We used puromycin-associated nascent chain proteomics (PUNCH-P) to characterize new protein synthesis in C2C12 myotubes and changes resulting from their exposure to the inflammatory mediators lipopolysaccharide (LPS) and interferon (IFN)-γ for either a short (4 h) or prolonged (16 h) time period. We identified sequences of nascent polypeptide chains belonging to a total of 1523 proteins and report their detection from three independent samples of each condition at each time point. The identified nascent proteins correspond to approximately 15% of presently known proteins in C2C12 myotubes and are enriched in specific cellular components and pathways. A subset of these proteins was identified only in treated samples and has functional characteristics consistent with the synthesis of specific new proteins in response to LPS/IFNγ. Thus, the identification of proteins from their nascent polypeptide chains provides a resource to analyze the role of new synthesis of proteins in both protein homeostasis and in proteome responses to stimuli in C2C12 myotubes. Our results reveal a profile of actively translating proteins for specific cellular components and biological processes in normal C2C12 myotubes and a different enrichment of proteins in response to LPS/IFNγ. Collectively, our data disclose a highly interconnected network that integrates the regulation of cellular proteostasis and reveal a diverse immune response to inflammation in muscle which may underlie the concomitantly observed atrophy and be important in inter-organ communication.

Lipoxygenase catalyzed metabolites derived from docosahexaenoic acid are promising antitumor agents against breast cancer.

Docosahexaenoic acid (DHA) is known to inhibit breast cancer in the rat. Here we investigated whether DHA itself or select metabolites can account for its antitumor action. We focused on metabolites derived from the lipoxygenase (LOX) pathway since we previously showed that they were superior anti-proliferating agents compared to DHA; 4-OXO-DHA was the most potent. A lipidomics approach detected several LOX-metabolites in plasma and the mammary gland in rats fed DHA; we also identified for the first time, 4-OXO-DHA in rat plasma. In a reporter assay, 4-OXO-DHA and 4-HDHA were more effective activators of PPARÉ£ than DHA. In breast cancer cell lines, 4-OXO-DHA induced PPARÉ£ and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) but inhibited the activity of NF-κB and suppressed PI3K and mTOR signaling. Because of the structural characteristics of 4-OXO-DHA (Michael acceptor), not shared by any of the other hydroxylated-DHA, we used MS and showed that it can covalently modify the cysteine residue of NF-κB. We have also shown that the chemopreventive effect of DHA is associated with significant reduction of PGE2 levels, in both rat mammary tumors induced by MNU and non-involved mammary tissues. Collectively, our results indicate that 4-OXO-DHA is the metabolite of choice in future chemoprevention studies.

The Ubiquitin-Proteasome Pathway and Epigenetic Modifications in Cancer.

BACKGROUND: The ubiquitin-proteasome pathway is involved in almost all cellular processes (cell cycle, gene transcription and translation, cell survival and apoptosis, cell metabolism and protein quality control) mainly through the specific degradation of the majority of intracellular proteins (>80%) or partial processing of transcription factors (e.g., NF-κB). A growing amount of evidence now indicates that epigenetic changes are also regulated by the ubiquitin-proteasome pathway. Recent studies indicate that epigenetic regulations are equally crucial for almost all biological processes as well as for pathological conditions such as tumorigenesis, as compared to non-epigenetic control mechanisms (i.e., genetic alterations or classical signal transduction pathways). OBJECTIVE: Here, we reviewed the recent work highlighting the interaction of the ubiquitin-proteasome pathway components (e.g., ubiquitin, E1, E2 and E3 enzymes and 26S proteasome) with epigenetic regulators (histone deacetylases, histone acetyltransferases and DNA methyltransferases). RESULTS: Alterations in the regulation of the ubiquitin-proteasome pathway have been discovered in many pathological conditions. For example, a 2- to 32-fold increase in proteasomal activity and/or subunits has been noted in primary breast cancer cells. Although proteasome inhibitors have been successfully applied in the treatment of hematological malignancies (e.g., multiple myeloma), the clinical efficacy of the proteasomal inhibition is limited in solid cancers. Interestingly, recent studies show that the ubiquitin-proteasome and epigenetic pathways intersect in a number of ways through the regulation of epigenetic marks (i.e., acetylation, methylation and ubiquitylation). CONCLUSION: It is therefore believed that novel treatment strategies involving new generation ubiquitinproteasome pathway inhibitors combined with DNA methyltransferase, histone deacetylase or histone acetyltransferase inhibitors may produce more effective results with fewer adverse effects in cancer treatment as compared to standard chemotherapeutics in hematological as well as solid cancers.

Omega-3 Fatty Acids Responsive Proteins and Reduction in Breast Density in Obese Postmenopausal Women.

We reported that breast density (BD) was inversely correlated with the plasma level of DHA in postmenopausal obese, but not in nonobese, women given Lovaza (n-3FA). To identify protein biomarkers for the possible differential effect of n-3FA on BD between obese and nonobese women, an iTRAQ method was performed to analyze plasma from obese and lean women at each time point (baseline, 12 and 24-months, n = 10 per group); 173 proteins with >95% confidence (Unuses Score >1.3 and local false discovery rate estimation <5%) were identified. Comparative analysis between various groups identified several differentially expressed proteins (hemopexin precursor, vitamin D binding protein isoform 1 precursor [VDBP], fibronectin isoform 10 precursor [FN], and α-2 macroglobulin precursor [A2M]). Western blot analysis was performed to verify the differential expression of proteins in the iTRAQ study, and those found to be altered in a tumor protective fashion by an n-3FA rich diet in our previous preclinical study; gelsolin, VDBP, and FN were altered by n-3FA in a manner consistent with reduction in inflammation in obese women. To test the impact of our findings on breast cancer risk reduction by n-3FA, a posthoc analysis revealed that n-3FA administration reduced BD selectively in obese postmenopausal women.

Survival Benefit Persists With Delayed Initiation of Adjuvant Chemotherapy Following Radical Cystectomy for Locally Advanced Bladder Cancer.

OBJECTIVE: To determine if delaying the initiation of adjuvant chemotherapy following radical cystectomy for locally advanced bladder cancer worsens overall survival. METHODS: This is a retrospective cohort study utilizing the National Cancer Database from 2006 to 2013. We included treatment-naïve patients who underwent radical cystectomy for muscle-invasive bladder cancer found to have locally advanced disease (pT3-T4 and/or pN+). Patients received no chemotherapy or multiagent adjuvant chemotherapy between 30 and 180 days following surgery. We used a multivariable Cox Regression to assess for differences in overall survival according to when patients initiated adjuvant chemotherapy. RESULTS: We identified 3590 patients: 2581 received no chemotherapy and 1009 received multiagent adjuvant chemotherapy. Adjuvant chemotherapy began 31-60 days postsurgery in 538 patients, 61-90 days in 321 patients, and 91-180 days in 150 patients. Relative to patients who did not receive chemotherapy, adjuvant chemotherapy decreased mortality when started 31-60 days (hazard ratio [HR], 0.60; 95% confidence interval [CI], 0.52-0.69; P <.001), 61-90 days (HR, 0.62; 95% CI, 0.53-0.74; P <.001), and 91-180 days following radical cystectomy (HR, 0.69; 95% CI, 0.55-0.87; P = .002). CONCLUSION: Adjuvant chemotherapy offers a survival benefit when started up to 6 months after radical cystectomy in patients with high-risk disease who did not receive neoadjuvant chemotherapy. Patients who require delayed initiation of adjuvant chemotherapy can still benefit from treatment.

Impact of Neoadjuvant Chemotherapy on Concordance of PD-L1 Staining Fidelity between the Primary Tumor and Lymph Node Metastases in Bladder Cancer.

OBJECTIVE: To evaluate programmed death ligand 1 (PD-L1) staining fidelity between the primary tumor and associated lymph node metastases in bladder cancer. To secondarily evaluate whether neoadjuvant chemotherapy (NAC) affects this relationship. METHODS: Sixty-seven subjects with residual bladder cancer on cystectomy and associated positive lymph nodes were identified between 2008 and 2015. PD-L1 staining of tumor cells was evaluated using H score and 49 specimens were also evaluated using combined positive score (CPS). Univariable and multivariable logistic regression analysis were used to assess how various clinical variables affected odds of PD-L1 fidelity between primary and metastatic tumors. RESULTS: Tumor PD-L1 staining was concordant in 79.1% of cases and CPS was concordant in 79.6% of cases. NAC did not significantly impact odds of PD-L1 or CPS fidelity (OR 1.974, 95% CI 0.673-5.784, OR 0.500, 95% CI 0.093-2.700). Among clinical variables analyzed on univariable analysis of tumor PD-L1 fidelity, H-score, and PD-L1 staining intensity were associated with significantly increased odds of PD-L1 fidelity and the association with staining intensity was confirmed on multivariable analysis. CONCLUSION: PD-L1 fidelity between primary bladder tumors and nodal metastases was observed in >75% of cases in this study. Additionally, NAC was not shown to diminish this propensity to maintain PD-L1 staining status. Further standardization of immunohistochemistry of tumor and infiltrating imsmune cells in metastatic bladder cancer is needed to improve application of therapeutics.

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.

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.

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.

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1.

BACKGROUND: The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. RESULTS: Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type, but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. CONCLUSIONS: Our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.

Radiation-Induced Alteration of the Brain Proteome: Understanding the Role of the Sirtuin 2 Deacetylase in a Murine Model.

Whole brain radiotherapy (WBRT) produces unwanted sequelae, albeit via unknown mechanisms. A deacetylase expressed in the central nervous system, Sirtuin 2 (SIRT2), has been linked to neurodegeneration. Therefore, we sought to challenge the notion that a single disease pathway is responsible for radiation-induced brain injury in Sirt2 wild-type (WT) and knockout (KO) mice at the proteomic level. We utilized isobaric tag for relative and absolute quantitation to analyze brain homogenates from Sirt2 WT and KO mice with and without WBRT. Selected proteins were independently verified, followed by ingenuity pathway analysis. Canonical pathways for Huntington's, Parkinson's, and Alzheimer's were acutely affected by radiation within 72 h of treatment. Although loss of Sirt2 preferentially affected both Huntington's and Parkinson's pathways, WBRT most significantly affected Huntington's-related proteins in the absence of Sirt2. Identical protein expression patterns were identified in Mog following WBRT in both Sirt2 WT and KO mice, revealing a proteomic radiation signature; however, long-term radiation effects were found to be associated with altered levels of a small number of key neurodegeneration-related proteins, identified as Mapt, Mog, Snap25, and Dnm1. Together, these data demonstrate the principle that the presence of Sirt2 can have significant effects on the brain proteome and its response to ionizing radiation.

Integrated quantitative analysis of nitrogen stress response in Chlamydomonas reinhardtii using metabolite and protein profiling.

Nitrogen starvation induces a global stress response in microalgae that results in the accumulation of lipids as a potential source of biofuel. Using GC-MS-based metabolite and iTRAQ-labeled protein profiling, we examined and correlated the metabolic and proteomic response of Chlamydomonas reinhardtii under nitrogen stress. Key amino acids and metabolites involved in nitrogen sparing pathways, methyl group transfer reactions, and energy production were decreased in abundance, whereas certain fatty acids, citric acid, methionine, citramalic acid, triethanolamine, nicotianamine, trehalose, and sorbitol were increased in abundance. Proteins involved in nitrogen assimilation, amino acid metabolism, oxidative phosphorylation, glycolysis, TCA cycle, starch, and lipid metabolism were elevated compared with nonstressed cultures. In contrast, the enzymes of the glyoxylate cycle, one carbon metabolism, pentose phosphate pathway, the Calvin cycle, photosynthetic and light harvesting complex, and ribosomes were reduced. A noteworthy observation was that citrate accumulated during nitrogen stress coordinate with alterations in the enzymes that produce or utilize this metabolite, demonstrating the value of comparing protein and metabolite profiles to understand complex patterns of metabolic flow. Thus, the current study provides unique insight into the global metabolic adjustments leading to lipid storage during N starvation for application toward advanced biofuel production technologies.

Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae.

BACKGROUND: Protein secretion is a fundamental process in all living cells. Proteins can either be secreted via the classical or non-classical pathways. In Saccharomyces cerevisiae, gluconeogenic enzymes are in the extracellular fraction/periplasm when cells are grown in media containing low glucose. Following a transfer of cells to high glucose media, their levels in the extracellular fraction are reduced rapidly. We hypothesized that changes in the secretome were not restricted to gluconeogenic enzymes. The goal of the current study was to use a proteomic approach to identify extracellular proteins whose levels changed when cells were transferred from low to high glucose media. RESULTS: We performed two iTRAQ experiments and identified 347 proteins that were present in the extracellular fraction including metabolic enzymes, proteins involved in oxidative stress, protein folding, and proteins with unknown functions. Most of these proteins did not contain typical ER-Golgi signal sequences. Moreover, levels of many of these proteins decreased upon a transfer of cells from media containing low to high glucose media. Using an extraction procedure and Western blotting, we confirmed that the metabolic enzymes (glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, glucose-6-phosphate dehydrogenase, pyruvate decarboxylase), proteins involved in oxidative stress (superoxide dismutase and thioredoxin), and heat shock proteins (Ssa1p, Hsc82p, and Hsp104p) were in the extracellular fraction during growth in low glucose and that the levels of these extracellular proteins were reduced when cells were transferred to media containing high glucose. These proteins were associated with membranes in vesicle-enriched fraction. We also showed that small vesicles were present in the extracellular fraction in cells grown in low glucose. Following a transfer from low to high glucose media for 30 minutes, 98% of these vesicles disappeared from the extracellular fraction. CONCLUSIONS: Our data indicate that transferring cells from low to high glucose media induces a rapid decline in levels of a large number of extracellular proteins and the disappearance of small vesicles from the extracellular fraction. Therefore, we conclude that the secretome undergoes dynamic changes during transition from glucose-deficient to glucose-rich media. Most of these extracellular proteins do not contain typical ER signal sequences, suggesting that they are secreted via the non-classical pathway.

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.

Survival during long-term starvation: global proteomics analysis of Geobacter sulfurreducens under prolonged electron-acceptor limitation.

The bioavailability of terminal electron acceptors (TEAs) and other substrates affects the efficiency of subsurface bioremediation. While it is often argued that microorganisms exist under "feast or famine", in the laboratory most organisms are studied under "feast" conditions, whereas they typically encounter "famine" in nature. The work described here aims to understand the survival strategies of the anaerobe Geobacter sulfurreduces under TEA-starvation conditions. Cultures were starved for TEA and at various times sampled to perform global comparative proteomic analysis using iTRAQ to obtain insight into the dynamics of change in proteins/enzymes expression associated with change in nutrient availability/environmental stress. Proteins varying in abundance with a high level of statistical significance (p < 0.05) were identified to understand how cells change from midlog to (i) stationary phase and (ii) conditions of prolonged starvation (survival phase). The most highly represented and significantly up-regulated proteins in the survival phase cells are involved in energy metabolism, cell envelope, and transport and binding functional categories. The majority of the proteins were predicted to be localized in the cell membranes. These results document that changes in the outer and cytoplasmic membranes are needed for survival of Geobacter under starvation conditions. The cell shuts down anabolic processes and becomes poised, through changes in its membrane proteins, to sense nutrients in the environment, to transport nutrients into the cell, and to detect or utilize TEAs that are encountered. Under TEA-limiting conditions, the cells turned from translucent white to red in color, indicating higher heme content. The increase in heme content supported proteomics results showing an increase in the number of cytochromes involved in membrane electron transport during the survival phase. The cell is also highly reduced with minimal change in energy charge (ATP to total adenine nucleotide ratio). Nonetheless, these proteomic and biochemical results indicate that even under TEA starvation cells remain poised for bioremediation.

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.