The Application of DNA Barcodes for the Identification of Marine Crustaceans from the North Sea and Adjacent Regions

PubMed Central

Raupach, Michael J.; Barco, Andrea; Steinke, Dirk; Beermann, Jan; Laakmann, Silke; Mohrbeck, Inga; Neumann, Hermann; Kihara, Terue C.; Pointner, Karin; Radulovici, Adriana; Segelken-Voigt, Alexandra; Wesse, Christina; Knebelsberger, Thomas

2015-01-01

During the last years DNA barcoding has become a popular method of choice for molecular specimen identification. Here we present a comprehensive DNA barcode library of various crustacean taxa found in the North Sea, one of the most extensively studied marine regions of the world. Our data set includes 1,332 barcodes covering 205 species, including taxa of the Amphipoda, Copepoda, Decapoda, Isopoda, Thecostraca, and others. This dataset represents the most extensive DNA barcode library of the Crustacea in terms of species number to date. By using the Barcode of Life Data Systems (BOLD), unique BINs were identified for 198 (96.6%) of the analyzed species. Six species were characterized by two BINs (2.9%), and three BINs were found for the amphipod species Gammarus salinus Spooner, 1947 (0.4%). Intraspecific distances with values higher than 2.2% were revealed for 13 species (6.3%). Exceptionally high distances of up to 14.87% between two distinct but monophyletic clusters were found for the parasitic copepod Caligus elongatus Nordmann, 1832, supporting the results of previous studies that indicated the existence of an overlooked sea louse species. In contrast to these high distances, haplotype-sharing was observed for two decapod spider crab species, Macropodia parva Van Noort & Adema, 1985 and Macropodia rostrata (Linnaeus, 1761), underlining the need for a taxonomic revision of both species. Summarizing the results, our study confirms the application of DNA barcodes as highly effective identification system for the analyzed marine crustaceans of the North Sea and represents an important milestone for modern biodiversity assessment studies using barcode sequences. PMID:26417993

Use of ethnic spices by adults in the United States: An exploratory study.

PubMed

Isbill, Jonathan; Kandiah, Jayanthi; Khubchandani, Jagdish

2018-01-01

Background: The use of complementary and alternative medicine (CAM) therapies has increased in the United States, but little is known about consumers' perceptions of use of such therapies. The purpose of this study was to assess knowledge, perceptions, and predictors of spice use for health promotion among adults in the Midwestern US. Methods: UUsing a cross-sectional study design, adults in the Midwestern US (n = 703) completed a valid and reliable survey which was pilot tested with a small convenience sample of adults (n = 38). The study variables included demographic profile, spice use behavior, perceptions about efficacy of spices, and willingness to use spices. Data were analyzed using SPSS to compute descriptive (e.g. percent and frequencies) and inferential statistics (i.e. logistic regression analyses). Results: Almost half of the participants were interested in learning about health benefits of spices (48%), indicated friends and family members as sources of information on spices (50%),and were willing to use spices as CAM therapies (51%). Most (>50%) of the participants were familiar with or had used eight out of the 10 listed spices. The majority of participants (54%)were currently using one or more spices on a daily basis and believed that ginger (64%), garlic(58%), and cinnamon (56%) could promote good health and wellness. In logistic regression analysis, age, gender (odds ratios [OR] = 1.44 and OR = 1.56), income (OR = 1.77), health status(OR = 2.01), and recommendations from healthcare providers (OR = 5.31 and OR = 3.96) were significant predictors of current spice use and willingness to use spices. Conclusion: Individuals in our study did not use many ethnic spices and were unaware of potential health benefits of spices. Greater awareness of ethnic spices for disease prevention and health promotion are needed in this population.

Use of ethnic spices by adults in the United States: An exploratory study

PubMed Central

Isbill, Jonathan; Kandiah, Jayanthi; Khubchandani, Jagdish

2018-01-01

Background: The use of complementary and alternative medicine (CAM) therapies has increased in the United States, but little is known about consumers’ perceptions of use of such therapies. The purpose of this study was to assess knowledge, perceptions, and predictors of spice use for health promotion among adults in the Midwestern US. Methods: UUsing a cross-sectional study design, adults in the Midwestern US (n = 703) completed a valid and reliable survey which was pilot tested with a small convenience sample of adults (n = 38). The study variables included demographic profile, spice use behavior, perceptions about efficacy of spices, and willingness to use spices. Data were analyzed using SPSS to compute descriptive (e.g. percent and frequencies) and inferential statistics (i.e. logistic regression analyses). Results: Almost half of the participants were interested in learning about health benefits of spices (48%), indicated friends and family members as sources of information on spices (50%),and were willing to use spices as CAM therapies (51%). Most (>50%) of the participants were familiar with or had used eight out of the 10 listed spices. The majority of participants (54%)were currently using one or more spices on a daily basis and believed that ginger (64%), garlic(58%), and cinnamon (56%) could promote good health and wellness. In logistic regression analysis, age, gender (odds ratios [OR] = 1.44 and OR = 1.56), income (OR = 1.77), health status(OR = 2.01), and recommendations from healthcare providers (OR = 5.31 and OR = 3.96) were significant predictors of current spice use and willingness to use spices. Conclusion: Individuals in our study did not use many ethnic spices and were unaware of potential health benefits of spices. Greater awareness of ethnic spices for disease prevention and health promotion are needed in this population. PMID:29423360

Development of utricular otoliths, but not saccular otoliths, is necessary for vestibular function and survival in zebrafish

NASA Technical Reports Server (NTRS)

Riley, B. B.; Moorman, S. J.

2000-01-01

We have been studying the consequences of embryonic vestibular dysfunction caused by the monolith (mnl) mutation in zebrafish. mnl is a dominant mutation that specifically inhibits formation of utricular otoliths. However, briefly immobilizing mnl/mnl embryos in agarose with the otic vesicle orientated at certain angles selectively induces or prevents formation of utricular and/or saccular otoliths. With this noninvasive technique, we generated six phenotypic classes of mnl/mnl mutants, designated S-S, U-U, U-S, S-US, U-US, and US-US, depending on which otoliths are present on each side (U, utricular otolith; S, saccular otolith). All mnl/mnl larvae survived through day 10 of development. Thereafter, S-S larvae showed a rapid decline, probably because of starvation, and none survived to adulthood. Survival rates in all other classes of mnl/mnl larvae (those having at least one utricular otolith) were close to normal. The presence or absence of utricular otoliths also correlated with vestibular function during early larval development, as measured by three criteria: First, unlike wild-type larvae, S-S mutant larvae showed almost no detectable counter-rotation of the eyes when tilted tail up or tail down. Second, 95% of S-S mutant larvae never acquired the ability to maintain a balanced dorsal-up posture. Third, although most wild-type larvae responded to gentle prodding by swimming in a straight line, S-S larvae responded by swimming in rapid circles, showing sudden and frequent changes in direction ("zigzagging"), and/or rolling and spiraling. All other phenotypic classes of mnl/mnl larvae behaved normally in these assays. These data demonstrate that bilateral loss of utricular otoliths disrupts the ability to sense gravity, severely impairs balance and motor coordination, and is invariably lethal. The presence of a utricular otolith in at least one inner ear is necessary and sufficient for vestibular function and survival. In contrast, saccular otoliths are dispensable for these functions. Copyright 2000 John Wiley & Sons, Inc.

Adherence to Web-Based Self-Assessments in Long-Term Direct-to-Patient Research: Two-Year Study of Multiple Sclerosis Patients.

PubMed

Jongen, Peter Joseph; Kremer, Ingrid E H; Hristodorova, Elena; Evers, Silvia M A A; Kool, Anton; van Noort, Esther M; Hiligsmann, Mickaël

2017-07-21

Direct-to-patient research via Web-based questionnaires is increasingly being used. Missed data or delayed reporting of data may negatively affect the quality of study results. It is insufficiently known to what degree patients adhere to agreed self-assessment schedule over the long term and whether questionnaires are filled out in a timely manner. The objective of this study was to investigate patients' adherence to a self-assessment schedule with low-frequency long questionnaires versus that with a high-frequency short questionnaire. In this study, the 36-item MS Impact Profile (MSIP) questionnaire measured (perceived) disabilities and the 54-item MS Quality of Life-54 (MSQoL-54) questionnaire measured health-related quality of life at 6-month intervals. Additionally, the 2-item Medication and Adherence (MA) questionnaire documented medication and adherence to disease-modifying medication every month. An experienced MS nurse assessed the Expanded Disability Status Scale (EDSS) score via phone. For both the self-assessment schedules, we calculated the percentage of patients who had completed all the questionnaires in the first 2 years (completion adherence), the percentage of patients who completed all the questionnaires within set time frames (interval adherence), the relationship between adherence and the EDSS score, and the timing of EDSS assessment. Of the 331 patients who enrolled themselves, 301 patients completed at least one questionnaire. At month six (M6), M12, M18, and M24, the MSIP was completed by 83.4% (251/301), 71.8% (216/301), 68.1% (205/301), and 58.5% (176/301) of the patients, respectively; the MSQoL-54 by 82.1% (247/301), 71.8% (216/301), 66.8% (201/301), and 57.1% (172/301), respectively; and the MA questionnaire by 80.1% (241/301), 70.4% (212/301), 62.1% (187/301), and 53.5% (161/301), respectively. For the MSIP, 56.8% (171/301) of the patients were 2-year completion adherent; 55.5% (167/301) and 53.5% (161/301) of the patients were completion adherent for the MSQoL-54 and MA questionnaires, respectively. Whereas 85.5% (142/166) of the patients were interval adherent for the MSIP and MSQoL-54, 25.5% (41/161) were interval adherent for the MA questionnaire, with 73.9% (119/161) exceeding the maximum MA monthly interassessment interval. Completion adherence for the monthly short MA questionnaire was higher in patients with moderately high disability (EDSS 5.0-5.5) than for those with no or minimal disability (EDSS 0-2.5) (OR 5.47, 95% CI 1.08-27.69; P=.040). Completion adherence was also higher in patients with EDSS assessment within 6 months after baseline than in those with later assessment (OR 1.810, 95% CI 0.999-3.280; P=.050). The 2-year completion adherence to Web-based self-assessments did not differ between the low-frequency long questionnaires and a high-frequency short questionnaire, but the interval adherence was substantially higher for the low-frequency long questionnaires. Personal contact with a member of the research team regarding a clinically relevant professional-reported outcome early in the study might positively affect the long-term completion adherence in direct-to-patient studies. ©Peter Joseph Jongen, Ingrid E.H. Kremer, Elena Hristodorova, Silvia M.A.A. Evers, Anton Kool, Esther M van Noort, Mickaël Hiligsmann. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 21.07.2017.

Evidence of sealing and brine distribution at grain boundaries in natural fine-grained Halite (Qum Kuh salt fountain, Central Iran): implications for rheology of salt extrusions

NASA Astrophysics Data System (ADS)

Desbois, Guillaume; Urai, Janos L.; de Bresser, J. H. P.

2010-05-01

When grain boundary movement is stopped, surface energy related forces reassert themselves driving the system to its equilibrium conditions ([2], [6], [7], [8]). This could result in growth of islands and shrinking of channels and hence in healing the boundary by internal redistribution of fluid and solid in the contact region. Such islands are proposed to grow preferentially close to the contact rim and promote the healing of the grain-grain contact, which in turn prevents transport in or out the boundary region and thus traps the fluids in isolated inclusions. This contribution is focused on observation of grain boundary microstructures in natural mylonitic rocksalt collected from the distal part of Kum-Quh salt fountain (central Iran) in order to give unprecedented insight of grain boundary microstructures using argon-beam cross-sectioning to prepare high quality polished surfaces suitable for high-resolution SEM imaging. The possibility to use our SEM under cryogenic conditions allows also imaging the in-situ distribution of fluids. Results show that brine at grain boundaries occurs as thick layers (> µm in scale) corresponding to cross-sectioned wetted triple junction tubes, as filling at triple junction and as array of isolated fluids inclusions at grain-grain contacts. Close observations at islands contacts suggest the presence of a very thin fluid film (<100 nm). The most remarkable is evidence for sealing of pore space appearing as subhedral crystals filling the void space and decoupled from surrounding crystals by a thin brine layer. In parallel to this microstructural study, we deformed the same samples in order to simulate the simple shear flow at very low mean stress as in the salt fountain. First results suggest a complicated rheology. Samples loaded at σ < 0.7 MPa show no measurable deformation in a month, indicating strain rates less than 10-12 s-1 though, in fully activated pressure-solution (PS) creep, strain rates of several orders of magnitude are expected for similar grain size ([5]). Other samples, which were loaded to 1 MPa before reducing the stress to 0.5 MPa deformed at much higher but variable rates, up to 10-8 s-1, in good agreement with activated PS creep. If, at first look, our pilot deformation experiments seem to reflect a kind of "yield stress" for activation PS creep ([7]); the experimental stress does not reach the theoritical condition to enable activation of PS. Thus, we interpret that the apparent "yielding stress" may not reflect strictu senso the "yielding stress" as described in [7] but rather to a "yielding stress" corresponding to the elastic reassessment of the grain system before the initiation of PS at privileged seal-brine-grain contacts. In salt fountain conditions, mylonitic samples are expected to be in the healing domain, but "jumps" in active stress required to activate PS creep is hardly probable. Thus, we suggest that rainwater influx plays a fundamental role in activation of PS. Rainwater should enable the marginal dissolution of healed contacts and then decreases in the area fraction of grain boundary occupied by solid island contact causing an increase in island stress. Therefore, this points to cyclic deformation of salt fountain: (1) during rainy periods the fountain will deformed at relative high strain rate by dominant PS; while (2) during dry seasons, it will not significantly flow because the grain boundary healing will prevent PS and lead to dominant dislocation creep. This interpretation is in good agreement with recent structural studies ([1], [4]), which gives evidence for both dynamic dislocation and pressure-solution creeps, and measurement of rapid flow after rainy periods with flow rates compatible with fully activated PS ([3]). [1] Desbois G., Zavada P., Schleder Z. and Urai J.L. (In review). Deformation and recrystallization mechanisms in naturally deformed salt fountain: microstructural evidence for a switch in deformation mechanisms with increased availability of meteoric water and decreased grain size (Qum Kuh, central Iran). Submitted to Journal of Structural Geology. [2] Ghoussoub J., and Leroy Y.M. (2001), Solid-fluid phase transformation within grain boundaries during compaction by pressure solution, J. Mech. Phys. Solids, 49, 737 2385-2430. 738 [3] Jackson, M.P.A., (1985). Natural strain in diapiric and glacial rock salt, with emphasis on Oakwood dome, East Texas, Bureau of Economic Geology, The University of Texas at Austin, Texas. [4] Schléder Z. and Urai J.L. (2007). Deformation and recrystallization mechanisms in mylonitic shear zones in naturally deformed extrusive Eocene-Oligocene rock salt from Eyvanekey plateau and Garmsar hills (central Iran). Journal of structural geology, 29, 241-255. [5] Spiers C.J. and Carter N.L. (1988). Microphysics of rock salt flow in nature. In: M. Aubertin and H.R. Hardy, Editors, The Mechanical Behaviour of Salt: Proceedings of the Fourth Conference Series on Rock and Soil Mechanics, TTP Trans Tech Publications, Clausthal-Zellerfeld, 22: 115-128. [6] Urai J. L. (1983). Water assisted dynamic recrystallization and weakening in polycrystalline bischofite. Tectonophysics 96 (1-2): 125-157. [7] Van Noort R., Visser H.J.M., Spiers C.J. (2008) Influence of grain boundary structure on dissolution controlled pressure solution and retarding effects of grain boundary healing. Journal of Geophysical Research, 113, B03201. [8] Visser, H. J. M. (1999). Mass transfer processes in crystalline aggregates containing a fluid phase, Ph. D. thesis, Utrecht University, Utrecht.

Poster Session B

PubMed Central

2014-01-01

B.1 Development of a Proximity Labeling Method to Identify the Protein Targets of Bioactive Small Molecules Zachary Hill, Min Zhuang, James Wells University of California, San Francisco, CA, USA Identifying the direct protein targets of a bioactive small molecule gives insight into the compound's mechanism of action, its efficacy, and possible toxicity. Target identification is becoming an increasingly important part of the drug- development process. However, given the transient and heterogeneous nature of interactions between small molecules and proteins, this step is often difficult, greatly slowing the development of new therapeutics. For this reason, new methods to rapidly identify the direct protein targets of bioactive small molecules are of great importance. Enrichment strategies coupled with quantitative mass spectrometry have shown great promise in target identification. Here we will present our progress toward developing an engineered enzymatic tagging method that enables specific labeling and enrichment of protein targets from complex lysates. This method couples the binding of a small molecule to a proximity-based labeling event. Labeled target proteins are enriched and subsequently identified using quantitative LC-MS/MS. We will discuss several variations of this method, and highlight our progress towards applying proximity labeling to small-molecule target identification and validation. B.2 Modelling Atherosclerosis: Molecular Changes in the Ascending Aorta of Cholesterol-fed Rabbits Jingshu Xu1,2, Mia Jüllig1,2, Martin J. Middleditch1,2, Garth J.S. Cooper1,2,3,4 1School of Biological Sciences, University of Auckland, New Zealand; 2Maurice Wilkins Centre for Molecular Biodiscovery, Faculty of Science, University of Auckland, New Zealand; 3Department of Pharmacology, Medical Sciences Division, University of Oxford, Oxford, UK; 4Centre for Advanced Discovery and Experimental Therapeutics, NIHR Manchester Biomedical Research Centre, the University of Manchester, Manchester, UK The cholesterol-fed rabbit is commonly used to study the effect of hypercholesterolaemia and the associated atherosclerotic lesions. Here we maintained New Zealand White rabbits on a diet containing 2% (w/w) cholesterol (HC diet) for 12 weeks, after which their ascending aortas were excised and subjected to proteomic analysis. Extracts from ten individually obtained ascending aorta samples were labelled with isobaric (iTRAQ) tags and analyzed by LC-MS/MS to profile the proteomic changes in response to the HC diet (n=5) in comparison with non-HC, standard diet (n=5). ProteinPilot was used to search the LC-MS/MS output against the NCBI rabbit protein sequence database, leading to identification of 453 unique proteins. Of these, 74 showed significant differences in relative abundance (p<0.05), with 69 proteins higher and five lower in ascending aorta from HC diet-fed rabbits compared to controls. Many of the observed protein changes are consistent with molecular perturbations within the ascending aorta in response to the HC diet in rabbits, e.g. elevation of apolipoproteins, extracellular matrix adhesion proteins, collagens, glycolytic enzymes, heat shock proteins, proteins involved in immune defence, and proteins regulating the polymeric state of actin. We also made a number of novel observations, including an extreme (16-fold) elevation of a protein previously linked to angiogenesis but not atherosclerosis. Numerous other proteins not previously associated with atherosclerosis were also increased in ascending aorta from HC-fed rabbits. These novel observations merit further investigation as these perturbations may play important and yet undiscovered roles in the pathogenesis of atherosclerosis. B.3 Post-translational Modification Networks Vera van Noort Katholieke Universiteit Leuven, Leuven, Belgium Protein post-translational modifications (PTMs) allow the cell to regulate protein activity and play a crucial role in the response to changes in external conditions or internal states. Advances in mass spectrometry now enable proteome wide characterization of PTMs and have revealed a broad functional role for a range of different types of modifications (1). We have systematically investigated the interplay of protein phosphorylation with other post-transcriptional regulatory mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae (2). Systematic perturbations by deletion of its only two protein kinases and its unique protein phosphatase identified not only the protein-specific effect on the phosphorylation network, but also a modulation of proteome abundance and lysine acetylation patterns, mostly in the absence of transcriptional changes. Reciprocally, deletion of the two putative N-acetyltransferases affects protein phosphorylation, confirming cross-talk between the two PTMs. The measured M. pneumoniae phosphoproteome and lysine acetylome revealed that both PTMs are very common, that (as in Eukaryotes) they often co-occur within the same protein and that they are frequently observed at interaction interfaces and in proteins that can be part of multiple protein complexes (3). The results imply previously unreported hidden layers of post-transcriptional regulation intertwining phosphorylation with lysine acetylation and other mechanisms that define the functional state of a cell. Aiming at a more global view of the interplay between PTM types, we collected modifications for 13 frequent PTM types in 8 eukaryotes, compared their speed of evolution and developed a method for measuring PTM co-evolution within proteins based on the co-occurrence of sites across eukaryotes (4). We found that PTM types are vastly interconnected, forming a global network that comprise in human alone >50,000 residues in about 6000 proteins. 1. Beltrao P, Bork P, Krogan NJ, van Noort V. Evolution and functional cross-talk of protein post-translational modifications. Mol Syst Biol 9, 714. (2013) 2. van Noort V, Seebacher J, Bader S, Mohammed S, Vonkova I, Betts MJ, Kühner S, Kumar R, Maier T, et al. Cross-talk between phosphorylation and lysine acetylation in a genome-reduced bacterium. Mol Syst Biol 8, 571. (2012) 3. Kühner S*, van Noort V*, etal. Proteome organization in a genome-reduced bacterium Science 326, 1235–1240. (2009) 4. Minguez P, Parca L, etal. Deciphering a global network of functionally associated post-translational modifications. Mol Syst Biol 8, 599. (2012) B.4 Extracellular Phosphorylation in the Murine Synaptosome Jonathan C Trinidad1, Ralf Schoepfer2, Alma L Burlingame3, Katalin F Medzihradszky3* 1Department of Chemistry, Indiana University, Bloomington, IN, USA; 2Department of Pharmacology, University College London, England, UK; 3Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, CA, USA Post-translational modifications (PTMs) play key regulatory roles in cellular localization and/or biological function of proteins. The site of modification within a protein; their fixed or transient nature; the stoichiometry and potential crosstalk between modifications have been the focus of numerous large-scale studies. Most of this research is focused on PTMs involved in intracellular processes, such as phosphorylation, methylation, GlcNAcylation, acetylation, and ubiquitination. Of these, phosphorylation is the most studied due to the biological importance of this modification as well as the availability of tools for such studies. While the majority of protein phosphorylation occurs on intracellular proteins, phosphorylation of secreted proteins is well established. Well-characterized examples include the secreted milk protein, beta-casein, and the serum protein fetuin. Our current understanding of the biological role of extracellular phosphorylation, as well as knowledge regarding the process by which they are modified, is incomplete. A kinase known as FAM20 has been identified and is localized to the Golgi as well as secreted. It has been shown to phosphorylate extracellular proteins with a SXE motif. We recently conducted extensive studies on the interplay between intracellular phosphorylation and GlcNAcylation on proteins isolated from murine synaptosomes. This data also allowed us to identify specific glycan structures on more than 500 secreted or transmembrane proteins. We have subsequently examined the extent to which phosphorylation was present in our sample on secreted proteins or extracellular regions of transmembrane proteins. Our goal was to determine the extent to which such phosphorylation can be explained by the known motif of Golgi-resident kinases. We analyzed the sequences around the extracellular phosphorylation sites, and the spatial relationship on the linear amino acid sequence between glycosylated and phosphorylated residues. This work was supported by NIH grant NIGMS 8P41GM103481, and by the Howard Hughes Medical Institute. B.5 Using Selective Reaction Monitoring (SRM) Mass Spectrometry To Unmask Regulatory Feedback Loops Controlling Adipogenesis Robert Ahrends1,2, Asuka Ota2, Kyle M. Kovary2, Takamasa Kudo2, Byung Ouk Park2, Mary N. Teruel2 1ISAS, Dortmund, Germany; 2Clinical and Systems Biology, Stanford University, Stanford, CA, USA Background: Due to modern lifestyle changes, obesity has a worldwide impact on human health. The obesity epidemic is now recognized as one of the most important public health problems facing the world today. Understanding adipogenesis is crucial to understanding obesity; failure of adipogenesis was shown to be a key factor in the development of diabetes. In earlier work using single-cell imaging, we demonstrated that there is a distinct decision made during the time course of adipogenesis. Thereby positive feedback loops between PPARg and other transcription factors (TFs) in the differentiation network are regulating this decision. We identified a positive feedback loop between PPARg and C/EBPb that plays a critical role in regulating adipogenesis. Since multiple feedback loops with different timing and strengths can sharpen the decision process and control the number of cells which are differentiating, we wanted to gain a better understanding of how many other proteins could be involved in the decision process. Objective: The objective of this work is to search for feedback loops that could play a key role in the commitment decision. Methods: Using Selected Reaction Monitoring (SRM) mass spectrometry combined with perturbations, we analyzed OP9 cells to detect peptides of TFs which can serve as probes. We validated these probes with isotopically coded internal peptide standards and established a SRM library of transcriptional key regulators. These probes were subsequently used to quantitatively profile different stages of adipogenesis to obtain time courses of different TFs. To achieve our major goal to elucidate the TF control network in more detail, we furthermore searched for hidden feedback loops in this differentiation system. To do so we chemically manipulated the activity level of PPARg and its potential feedback partners individually. If a protein was a component of one or several feedback loops and was experimentally manipulated, all the other components of feedback loops associated with this protein should display a relative change in abundance and vice versa. Results: We developed a SRM methodology to monitor the concentration changes of TFs during adipogenesis. Using this SRM methodology together the perturbation of PPARg, and single cell analysis we were able to validate known feedback loops (C/EBPa, C/EBPb) and to identify several new feedback Loops. Conclusions: PPARg is the master regulator of adipogenesis. To successfully differentiate preadipocytes into adipocytes, its activity needs to be tightly regulated by a network of feedback loops. Overall, the study provides a new SRM MS-based method to uncover novel feedback loops regulating TFs. Based on this method; we have identified 7 new proteins which are fundamental regulators of PPARg and the fat cell commitment decision. B.6 Application of Quantitative and Functional Phosphoproteomics In Study of Ethylene Signaling Ning Li 1 1The Hong Kong University of Science and Technology, Hong Kong, China Ethylene is a major plant hormone that regulates a diverse aspect of plant growth and development. The regulatory roles of ethylene in plants include promotion of leaf and flower petal senescence, yellowing and abscission, as well as promotion of fruit abscission and ripening. This key hormone is also involved in regulation of a number of plant biotic and abiotic stress responses. A dramatic effect of ethylene on tropic response is the dual-and-opposing effect of ethylene on stem negative gravitropic response, in which short-term ethylene treatment (0.5 hour) appears to inhibit stem bending up following re-orientation of inflorescence of Arabidopsis. In contrast, a long-term treatment (12 hours) stimulates gravitropic response and promote stem curve up faster. This time-dependent and dose- independent dual-and-opposing effect of ethylene on stem gravitropism may involve multiple signaling pathways. Stable isotope metabolic labeling-based quantitative phosphoproteomics performed on ein2–5, ctr1–1 and rcn1–1 ethylene signaling mutants indeed confirmed the time-dependent protein phosphorylation changes and some of phosphorylation events are independent to ein2 loss-of- function gene in response to ethylene treatment. Functional studies on the phosphorylated transcription factor ERF110 isoform suggest that it is required for the control of flowering time via multiple ethylene signaling pathways. B.7 Intact N- and O-linked Glycopeptide Identification from HCD Data Using Byonic Katalin F. Medzihradszky1, Jason Maynard1, Krista Kaasik1, Marshall Bern2 1University of California, San Francisco, CA, USA; 2Protein Metrics, San Carlos, CA, USA The importance of high quality analysis of glycosylated proteins is steadily increasing. Both the regulatory and signaling functions of the intracellular GlcNAc modification are widely documented, and different enrichment strategies for GlcNAcylated peptides have been developed. Extracellular glycosylation has been linked to a wide variety of diseases and both N- and O-glycosylation play important roles in providing the structural integrity of certain proteins, controlling protein clearance, protein-protein interaction, and enzymatic processing. Furthermore, most protein pharmaceuticals are glycosylated, and thus, batch to batch characterization of these drugs also involves glycosylation analysis. Towards the end of the last century mass spectrometry has become the method of choice for the analysis of post-translational modifications, and high throughput workflows have been developed for a number of different PTMs. Though mass spectrometry has been used for the characterization of N- and O-glycosylation of single proteins, its high-throughput application was prevented by a number of issues. These issues include the non-pattern based complex oligosaccharide structures, the isomeric building blocks, as well as the extensive carbohydrate fragmentation upon collisional activation. In-depth carbohydrate analysis still requires different tools: the released glycan pools are studied derivatized or unmodified using a wide variety of analytical methods, such as capillary electrophoresis, chromatography, exoglycosidase cocktails, and NMR. While such techniques will provide information on the identity of the sugar units and their linkages, the information on the localization of glycans within the protein sequence and of their site-specific heterogeneity is lost. Electron-transfer dissociation, which preserves peptide side-chain modifications, has enabled the MS/MS analysis of intact glycopeptides, and led to the successful assignment of thousands of GlcNAcylated sequences and Golgi-derived glycopeptides. This gave a boost to intact glycopeptide analysis, and search engines such as Protein Prospector and Byonic can handle even complex glycan mixtures. The most recommended acquisition workflow uses the diagnostic HexNAc oxonium ion produced by HCD analysis to trigger ETD analysis. Unfortunately, because extracellular glycosylation increases the peptide mass significantly without additional charge added, glycopeptides frequently produce low charge-density precursor ions that will yield only charge-reduced molecules upon ETD activation. However, properly acquired HCD data may contain sufficient information for glycopeptide identification. Byonic has been adjusted for the interpretation of such spectra. We will present the results from a complex N- and O-linked glycopeptide-containing mixture isolated from mouse brain synaptosome using WGA lectin weak affinity chromatography, highlighting the advantages and limitations of this approach. This work was supported by NIH grant NIGMS 8P41GM103481, and by the Howard Hughes Medical Institute (to the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF, Director: A.L. Burlingame). B.8 Factors that Contribute to the Complexity of Glycopeptide Analysis – Besides Site-specific Heterogeneity Katalin F. Medzihradszky1, Zsuzsa Darula 1University of California, San Francisco, CA, USA; 2Laboratory of Proteomics Research, Biological Research Centre, Szeged, Hungary Data about the biological significance of extracellular glycosylation are compiling. Aberrant glycosylation has been implicated in diseases. Extracellular glycan remodeling has been linked to intracellular signaling. At the same time high-throughput intact glycopeptide analysis still is in its infancy. Glycosylation is a ‘special’ PTM. It includes a wide variety of structures formed by different biological pathways, performing very different biological functions. It is also unique in that aspect that the different MS/MS activation techniques provide different clues to solve the glycopeptide ‘puzzle’, but none delivers all the necessary information. Ion trap CID provides information about the glycan structure and usually reveals the mass of the peptide modified. Beam-type CID (HCD) frequently provides sufficient fragmentation information to assign the sequence modified. However, site assignment for O-glycopeptides is rarely possible. ETD identifies the sequence and site(s) modified, but one has to know the glycans present. Thus, using all these data together would offer the best solution. However, even the search engines aimed at glycopeptide identification cannot utilize all information available. We analyzed large and complex intact glycopeptide datasets, generated by LC/MS analysis of lectin-affinity-enriched mouse synaptosome and bovine or human serum samples. We will show that ETD data alone with faulty monoisotopic precursor peak-assignment, non-specific proteolytic digestion or covalent peptide modification will ‘assign’ an incorrect glycan structure. We will also present that a common buffer ingredient, Tris – widely used, even recommended for lectin-affinity chromatography- modifies sialic acid, altering not only its mass but also the chromatographic behavior of the glycopeptides. HCD analysis was instrumental in deciphering this unexpected side reaction. Acknowledgments – KFM was supported by NIH grant NIGMS 8P41GM103481, and by the Howard Hughes Medical Institute (to the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF, Director: A.L. Burlingame) and by the following grants: OTKA 105611 (to Z. Darula), and BAROSS-DA07-DA-ESZK-07–2008-0036 (to the Biological Research Centre, HAS, director: P. Ormos). Z. Darula was supported by the Janos Bolyai Fellowship of the HAS. B.9 Characterizing Qualitative and Quantitative Global Changes in the Aging Heart Using pSMART, a Novel Acquisition Method Maryann S. Vogelsang, Amol Prakas, David Sarracino, Gouri Vadali, Scott Peterman Thermo Fisher Scientific, BRIMS Center, Cambridge, MA, USA The cardiovascular system has been shown to undergo significant changes as it ages. These changes range from genomic to structural. We have completed a label-free quantitative global profiling and targeted analysis of the cardiac proteome in aging mice using a novel data acquisition method, pSMART. Heart tissue was isolated and homogenized from both young (2 months old) and old (2 years old) mice. Solubilized and digested protein samples were spiked with the PRTC peptide retention time trainer kit and analyzed using unbiased data-dependent acquisition (DDA) method. Initial characterization experiments using unbiased DDA facilitated the building of detailed murine cardiac tissue spectral library. The spectral library records contain the relative retention time information based on the standard peptides as well as highly confident endogenous peptides, precursor and product ion information such as measured mass values and relative abundance used to create a consensus product ion spectrum. The spectral library information was used to create reference information to perform qual/quan determination in real-time. The pSMART method was used to acquire qualitative/ quantitative data analysis using one HR/AM MS and a series of narrow DIA mass windows. Our pSMART strategy resulted in 30% more peptide identifications per run than a standard DDA run. Additionally, using pSMART, we were able to confirm MS1 quantitation at low abundance levels with MS/MS for each peptide. This novel acquisition enabled quantitation of previously identified peptides as well as novel putative targets of aging. By identifying and quantifying more targets, we were able to better characterize the dynamic proteomic changes of cardio-dysfunction in aging mice. B.10 Quantitative Site-Specific Profiling S-glutathionylation in Macrophages in Response to Engineered Nanomaterial-induced Oxidative Stress Jicheng Duan, Vamsi K. Kodali, Matthew J. Gaffrey, Jia Guo, Rosalie K. Chu, David G. Camp, Richard D. Smith, Brian Thrall, Wei-Jun Qian Pacific Northwest National Laboratory, Richland, WA, USA Engineered nanoparticles are emerging functional materials with unique physicochemical properties, which make them desirable for commercial and medical applications. It is important to assess the toxicity of nanomaterials and recognize the underlying mechanisms of their toxicity. Oxidative stress is known to play important roles in nanomaterial-induced cellular toxicity, which leads to the generation of reactive oxygen species and alteration of protein activities and functions in cells. However, the knowledge about proteins and signaling pathways associated with nanomaterial-induced oxidative stress and nanotoxicity is still limited. Reversible cysteine-based protein modifications, such as S-glutathionylation (SSG) and S-nitrosylation (SNO), represent an important mechanism that modulates diverse cellular pathways in response to the disturbance of redox balance in cells. These redox modifications would be a potential regulatory mechanism in response to nanomaterials-induced oxidative stress and nanotoxicity. Recently, we have developed an effective proteomic approach for site-specific identification and quantification of different cysteine-based redox modifications by integrating selective reduction of oxidized cysteines, resin-assisted enrichment of thiol-containing proteins, and isobaric labeling to enable LC-MS/MS-based quantification. Herein, we present the preliminary results about the alteration of protein SSG modifications in mouse macrophages after exposure to different nanoparticles (CoO, Fe3O4 and SiO2 nanoparticles) by quantitative site-specific profiling. We observed that among these nanoparticles, CoO nanoparticles led to the most significant dose-dependent cytotoxicity and increase of protein SSG modifications in macrophages. Our site-specific SSG data highlighted redox sensitive proteins and their specific Cys residues potentially implicated in oxidative stress response. Functional analysis revealed that the most significantly enriched molecular function categories for SSG-modified proteins were free radical scavenging and cell death/survival. This preliminary result provides some insights on protein SSG modification as a potential regulatory mechanism of nanomaterial-induced oxidative stress. B.11 O-GlcNAc Regulates SOX2 Activity in Embryonic Stem Cells by Altering Protein-SOX2 Interactions Samuel Myers1, Sailaja Pedadda, Tara Freidrich, Sean Thomas, Gregor Krings, Michael Lopez, Marena Trinidad, Barbara Panning, Al Burlingame University of California, San Francisco, CA, USA SOX2 is a versatile transcription factor that maintains embryonic stem cell (ESC) pluripotency and self-renewal, and is important for proper lineage specification and adult stem cell maintenance. This versatility is likely due to post-translational modifications (PTMs) as SOX2 has been reported to be modified by numerous chemical moieties in a variety of cell types. One such PTM is O-GlcNAc, the dynamic and regulatory glycosylation of intracellular proteins. Global O-GlcNAc is essential for ESC self-renewal though the function of SOX2 O-GlcNAcylation in ESC is not understood. Here, we show that SOX2 is O-GlcNAc modified in the transactivation domain and alterations of self-renewing signals induce changes in SOX2 O-GlcNAc stoichiometry. Replacement of wild-type SOX2 with an O-GlcNAc-deficient mutant SOX2 in ESCs increases the pluripotency transcriptional network while down-regulating genes involved in differentiation. Analysis of SOX2-interacting proteins from ESCs revealed that the WT and mutant SOX2 interact with distinct subsets of transcriptional regulatory complexes. Thus, SOX2 O-GlcNAcylation modulates the transcriptional landscape of ESCs by modulating SOX2 activity and interactions with epigenetic regulatory complexes. B.12 Development of Multiplexed Assays for Oral Cancer Biomarker Verification by Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Yung-Chin Hsiao1, Lang-Ming Chi2, Kun-Yi Chien1, Yi-Ting Chen1, Yu-Sun Chang1, Jau-Song Yu1 1Chang Gung University, Tao-Yuan, Taiwan; 2Chang Gung Memorial Hospital, Tao-Yuan, Taiwan Oral cancer, one of the common cancers in Taiwan and other areas of Southern Asia, has become an increasing burden on the health care system in this region. Although numerous potential oral cancer biomarkers have been discovered in the past decades, very few of them have been verified and validated in parallel to compare their clinical utility. Recently, a multiplexed, targeted proteomics assay platform, termed SISCAPA-MRM-MS (stable isotope standards and capture by anti-peptide antibodies combined with multiple reaction monitoring mass spectrometry) has been shown to be a feasible approach for verifying multiple protein biomarker candidates in body fluid samples. Therefore, we sought to prioritize biomarker candidates from published literature and our in-house database and develop a high-throughput/multiplexed SISCAPA-MRM-MS assay for quantifying potential oral cancer biomarker candidates. We produced ∼400 clones of anti-peptide mAbs against 50 selected targets and effectively sieved out the high quality anti-peptide mAbs against 24 targets according to their binding affinity to peptide antigens (using peptide-immobilized SPR system) and immuno-capture capability (using SISCAPA-MS assay). These mAbs were then assembled into a 24-plex SISCAPA-MRM MS assay and applied to preliminarily evaluation of these 24 candidates in pooled saliva samples obtained from oral cancer patients and healthy controls. Eight of the 24 candidates were found to be drastically increased in pooled saliva samples from oral cancer patients as compared with healthy controls. The promise of this 24-plex SISCAPA LC-MRM MS assay allows us to systematically evaluate the abundance of targets in clinical samples for oral cancer biomarker discovery in the near future. B.13 Characterisation of Glycosylation of Paramyxovirus Surface Glycoproteins by Mass Spectrometry Cassandra L. Pegg1, C. Hoogland1, S.M. Johnson2, C.C. Gonzalez2, M.E. Peeples2, J.J. Gorman1 1QIMR Berghofer Medical Research Institute, Herston, Australia; 2Center for Vaccines & Immunity, The Research Institute at Nationwide Children's Hospital, Columbus, OH, USA The family Paramyxoviridae (paramyxovirus) contains a number of significant human and animal pathogens. Represented within this family are human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV) and Newcastle disease virus (NDV). The former two cause severe respiratory tract disease in infants, children and immunocompromised individuals. At present, safe and effective vaccines are not available for hRSV and hMPV. NDV is the causative agent of Newcastle disease (ND) afflicting a wide range of avian species. The desire to study NDV is due not only to the significant economic impact it has on the poultry industry worldwide but also its potential use as an oncolytic agent and vaccine vector for human and animal use. Additionally, findings on NDV may be translated to closely related viruses that cause disease in humans, such as parainfluenza viruses. Of great importance to paramyxoviruses are the variable attachment glycoproteins, hemagglutinin (H), hemagglutinin-neuraminidase (HN) and major surface glycoprotein (G) along with the fusion (F) glycoprotein. Glycoproteins H, HN and G are involved in viral attachment to the host cell, while F is responsible for viral entry by means of fusion with host cell membranes. Research has shown that the glycosylation sites present on these proteins can modulate the ability of the virus to infect host cells and stimulate the host immune system. Characterisation of site-specific glycan heterogeneity remains one of the few unexplored areas related to hRSV, hMPV and NDV surface glycoproteins. Previous research has been conducted to determine glycan heterogeneity, but not glycan site specificity, of NDV F. As yet, glycan site occupancy and glycan heterogeneity for glycoproteins G and F of hRSV and hMPV and HN of NDV, have not been defined at a chemical level. Revealing the glycosylation profile of these proteins may help elucidate mechanisms of viral attachment, replication and immune evasion within paramyxoviruses. Additionally, accurate identification and characterisation of protein glycosylation is required for producing glycoprotein therapeutics and for the development of targeted treatments. Liquid chromatography-MS/MS strategies utilising ETD, HCD and CID fragmentation were implemented to structurally characterise the digested glycoproteins. Initial research has revealed complex N-linked and mucin-like O-linked glycosylation of recombinant RSV G. Analysis of NDV revealed high-mannose N-linked glycans of F glycoprotein as well as high mannose and sialylated and sulphated complex N-linked glycans and a novel sialylated O-linked glycan of NDV HN. B.14 Developing A New In Vivo Cross-linking Mass Spectrometry Platform to Define Protein-Protein Interactions in Living Cells Robyn M. Kaake1, Xiaorong Wang1, Anthony Burke1, Clinton Yu1, Wynne Kandur1, Yingying Yang1, Eric J. Novtisky1, Tonya Second2, Jicheng Duan1, Athit Kao1, Shenheng Guan3, Danielle Vellucci1, Scott D. Rychnovsky1, Lan Huang1 1University of California, Irvine, CA, USA; 2Thermo Fisher Scientific, Waltham, MA, USA; 3University of California, San Francisco, CA, USA Protein-protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane permeable, enrichable and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells, and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and at the targeted protein complex level. Our work represents a general approach in studying in vivo PPIs, and provides a solid foundation for future studies towards the complete mapping of PPI networks in living systems. B.15 High-resolution Orbitrap Characterization of Preferential Chain Pairing in Co-expressed Bispecific Antibody Production by MS Under Native and Acidic Conditions Luis Schachner, Jianhui Zhou, Luke McCarty, Diego Ellerman, Michael Dillon, Christoph Spiess, Jennie Lill, Paul Carter, Wendy Sandoval Departments of Protein Chemistry and Antibody Engineering, Genentech, Inc., South San Francisco, CA, USA Bispecific antibodies possess the characteristics and binding specificity of two distinct monoclonal antibodies, and as such can bind to two targets or epitopes simultaneously. Bispecific antibodies have recently received great attention for their promising results in clinical trials or potential new modes to deliver therapeutics. Generation of a bispecific antibody by co-expression of two light and heavy chains, would result in several mispaired species. While the “knobs-into-holes” technology enables efficient hetero-dimerization of the two heavy chains, the presumed random mispairing of the light chains has not been studied in detail as technologies to readily characterize and quantify the heterodimer species were missing. Using an anti-IL-4/IL-13, a bispecific antibody, which targets the IL-4 and IL-13 cytokines involved in type 2 cytokine-induced inflammation, we describe a mass spectrometry characterization assay under native and acidic conditions for co-expressed bispecific antibodies using an Exactive Plus Extended Mass Range (EMR) Orbitrap instrument. The high mass resolving power of the EMR Orbitrap allows unambiguous identification of all light and heavy chain pairing variants in a mixture of bispecific antibodies randomly assembled in vivo upon co-expression. Using the EMR Orbitrap technology, we identify and characterize the preferential pairing of the anti-IL-13 light chain to its cognate heavy chain. This unexpected, non-random pairing may be leveraged to guide the design of a single-cell solution for the production of bispecific antibodies. B.16 Controlling Low Rates of Cell Differentiation through Noise and Ultra-high Feedback Robert Ahrends, Asuka Ota, Kyle M. Kovary, Takamasa Kudo, Byung Ouk Park, Mary N. Teruel Dept. of Chemical and Systems Biology, Stanford University, Stanford, CA, USA The tissue size of adult mammals is maintained by replacement of aging or damaged cells by slow, ongoing cell differentiation. Disruption of this rate of ongoing differentiation results in serious disease. For example, acute myeloid leukaemia (AML) is caused by a block in differentiation which results in precursor cells proliferating uncontrollably rather than differentiating into a more terminal state. Adipocytes, the key regulators of glucose and lipid metabolism, make up 10–40% of human body mass and are renewed at a rate of approximately 10% per year [1]. The adipocyte system that will be used has the unique advantage that the terminal differentiation transition is relatively short and experimentally accessible using single cell microscopy. Understanding how cells regulate such very slow differentiation rates may enable better treatments of metabolic diseases and obesity. Here we combine quantitative mass spectrometry [2–4], computational modeling, and single-cell microscopy [5] to identify the network architecture that enables pre-adipocytes to differentiate at a rate of only 0.5% every 4 days. We show that that cell-to-cell variability, or noise, in protein abundance acts within a network of more than six positive feedbacks to permit pre-adipocytes to differentiate at very low rates. This system architecture resolves two fundamental opposing requirements: to irreversibly lock cells in the differentiated state and to create large cell-to-cell signal variability to enable differentiation at very low rates. The resolution of this optimization problem by noise and ultra-high feedback connectivity provides a generalizable mechanism for mammalian tissue size control. References: 1. [1] Spalding KL et al. (2008). Dynamics of fat cell turnover in humans. Nature. Jun 5; 453(7196):783–7. 2. [2] Abell E, Ahrends R, Bandara S, Park BO, Teruel MN. (2011). Parallel adaptive feedback enhances reliability of the Ca2+ signaling system. Proc Natl Acad Sci U S A. Aug 30; 108(35):14485–90. 3. [3] Ahrends R, Ota A, Kovary KM, Kudo T, Park BO, Teruel MN. (2014). Controlling low rates of cell differentiation through noise and ultra-high feedback. Science 345, June 20. 4. [4] Ota A, Kovary KM, Shen W, Ahrends R, Kraemer FB, Teruel MN. Using selective reaction monitoring (SRM) mass spectrometry to profile nuclear protein abundance differences between adipose tissue depots of insulin-resistant mice. (submitted) 5. [5] Park BO, Ahrends R, Teruel MN. (2012). Consecutive positive feedback loops create a bistable switch that controls preadipocyte to adipocyte conversion. Cell Reports Oct 25.