Database Search Strategies for Proteomic Data Sets Generated by Electron Capture Dissociation Mass Spectrometry

Large data sets of electron capture dissociation (ECD) mass spectra from proteomic experiments are rich in information; however, extracting that information in an optimal manner is not straightforward. Protein database search engines currently available are designed for low resolution CID data, from which Fourier transform ion cyclotron resonance (FT-ICR) ECD data differs significantly. ECD mass spectra contain both z-prime and z-dot fragment ions (and c-prime and c-dot); ECD mass spectra contain abundant peaks derived from neutral losses from charge-reduced precursor ions; FT-ICR ECD spectra are acquired with a larger precursor m/z isolation window than their low-resolution CID counterparts. Here, we consider three distinct stages of postacquisition analysis: (1) processing of ECD mass spectra prior to the database search; (2) the database search step itself and (3) postsearch processing of results. We demonstrate that each of these steps has an effect on the number of peptides identified, with the postsearch processing of results having the largest effect. We compare two commonly used search engines: Mascot and OMSSA. Using an ECD data set of modest size (3341 mass spectra) from a complex sample (mouse whole cell lysate), we demonstrate that search results can be improved from 630 identifications (19% identification success rate) to 1643 identifications (49% identification success rate). We focus in particular on improving identification rates for doubly charged precursors, which are typically low for ECD fragmentation. We compare our presearch processing algorithm with a similar algorithm recently developed for electron transfer dissociation (ETD) data

Living on the Margin in the Anthropocene: Engagement Arenas for Sustainability Research and Action at the Ocean-Land Interface

The advent of the Anthropocene underscores the need to develop and implement transformative governance strategies that safeguard the Earth\u27s life-support systems, most critically at the ocean-land interface - the Margin. The seaward realm of the Margin is the new frontier for resource exploitation and colonization to meet the needs of coastal nations and humanity overall. Here, we spotlight the pivotal role of the Margin for planetary resilience and sustainability, highlight priority issues, and outline a research strategy which aims to: (a) better understand Margin social-ecological systems; (b) guide sustainable development of Margin resources; (c) design governance regimes to reverse unsustainable practices; (d) facilitate equitable sharing of Margin resources; and (e) evaluate alternative research approaches and partnerships that address major Margin challenges. © 2015 The Authors

Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics

The addition of the monosaccharide beta-N-acetyl-D-glucosamine to proteins (O-GlcNAc glycosylation) is an intracellular, post-translational modification that shares features with phosphorylation. Understanding the cellular mechanisms and signaling pathways that regulate O-GlcNAc glycosylation has been challenging because of the difficulty of detecting and quantifying the modification. Here, we describe a new strategy for monitoring the dynamics of O-GlcNAc glycosylation using quantitative mass spectrometry-based proteomics. Our method, which we have termed quantitative isotopic and chemoenzymatic tagging (QUIC-Tag), combines selective, chemoenzymatic tagging of O-GlcNAc proteins with an efficient isotopic labeling strategy. Using the method, we detect changes in O-GlcNAc glycosylation on several proteins involved in the regulation of transcription and mRNA translocation. We also provide the first evidence that O-GlcNAc glycosylation is dynamically modulated by excitatory stimulation of the brain in vivo. Finally, we use electron-transfer dissociation mass spectrometry to identify exact sites of O-GlcNAc modification. Together, our studies suggest that O-GlcNAc glycosylation occurs reversibly in neurons and, akin to phosphorylation, may have important roles in mediating the communication between neurons

Demasculinization of male guppies increases resistance to a common and harmful ectoparasite

Parasites are detrimental to host fitness and therefore should strongly select for host defence mechanisms. Yet, hosts vary considerably in their observed parasite loads. One notable source of inter-individual variation in parasitism is host sex. Such variation could be caused by the immunomodulatory effects of gonadal steroids. Here we assess the influence of gonadal steroids on the ability of guppies (Poecilia reticulata) to defend themselves against a common and deleterious parasite (Gyrodactylus turnbulli). Adult male guppies underwent 31 days of artificial demasculinization with the androgen receptor-antagonist flutamide, or feminization with a combination of flutamide and the synthetic oestrogen 17 β-estradiol, and their parasite loads were compared over time to untreated males and females. Both demasculinized and feminized male guppies had lower G. turnbulli loads than the untreated males and females, but this effect appeared to be mainly the result of demasculinization, with feminization having no additional measurable effect. Furthermore, demasculinized males, feminized males and untreated females all suffered lower Gyrodactylus-induced mortality than untreated males. Together, these results suggest that androgens reduce the ability of guppies to control parasite loads, and modulate resistance to and survival from infection. We discuss the relevance of these findings for understanding constraints on the evolution of resistance in guppies and other vertebrates

Chemotaxis: a feedback-based computational model robustly predicts multiple aspects of real cell behaviour

The mechanism of eukaryotic chemotaxis remains unclear despite intensive study. The most frequently described mechanism acts through attractants causing actin polymerization, in turn leading to pseudopod formation and cell movement. We recently proposed an alternative mechanism, supported by several lines of data, in which pseudopods are made by a self-generated cycle. If chemoattractants are present, they modulate the cycle rather than directly causing actin polymerization. The aim of this work is to test the explanatory and predictive powers of such pseudopod-based models to predict the complex behaviour of cells in chemotaxis. We have now tested the effectiveness of this mechanism using a computational model of cell movement and chemotaxis based on pseudopod autocatalysis. The model reproduces a surprisingly wide range of existing data about cell movement and chemotaxis. It simulates cell polarization and persistence without stimuli and selection of accurate pseudopods when chemoattractant gradients are present. It predicts both bias of pseudopod position in low chemoattractant gradients and-unexpectedly-lateral pseudopod initiation in high gradients. To test the predictive ability of the model, we looked for untested and novel predictions. One prediction from the model is that the angle between successive pseudopods at the front of the cell will increase in proportion to the difference between the cell's direction and the direction of the gradient. We measured the angles between pseudopods in chemotaxing Dictyostelium cells under different conditions and found the results agreed with the model extremely well. Our model and data together suggest that in rapidly moving cells like Dictyostelium and neutrophils an intrinsic pseudopod cycle lies at the heart of cell motility. This implies that the mechanism behind chemotaxis relies on modification of intrinsic pseudopod behaviour, more than generation of new pseudopods or actin polymerization by chemoattractant

An evaluation of the Weldon Spring Feed Preparation and Sampling Plant

A description of the new Weldon Spring Feed Preparation and Sampling Plant for uranium concentrates is given. Prior to the startup of this plant the auger to be later installed was used in an evaluation program to test reliability for representative sampling and uniformity both within drums and between drums of various concentrates. Results of this program were used as a reference for the sampling plant evaluation which involved successive auger and mechanical sampling of a series of lots of several concentrates, followed by moisture determinations, uranium assays, and statistical analyses of the data. From the final results conclusions are drawn concerning the suitability of the mechanical sampling system for the concentrates examined

SILAC-based proteomic quantification of chemoattractant-induced cytoskeleton dynamics on a second to minute timescale

Cytoskeletal dynamics during cell behaviours ranging from endocytosis and exocytosis to cell division and movement is controlled by a complex network of signalling pathways, the full details of which are as yet unresolved. Here we show that SILAC-based proteomic methods can be used to characterize the rapid chemoattractant-induced dynamic changes in the actin–myosin cytoskeleton and regulatory elements on a proteome-wide scale with a second to minute timescale resolution. This approach provides novel insights in the ensemble kinetics of key cytoskeletal constituents and association of known and novel identified binding proteins. We validate the proteomic data by detailed microscopy-based analysis of in vivo translocation dynamics for key signalling factors. This rapid large-scale proteomic approach may be applied to other situations where highly dynamic changes in complex cellular compartments are expected to play a key role