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

The Social and Economic Long Term Monitoring Program (SELTMP) 2014: Recreation in the Great Barrier Reef

[Extract] Introduction.\ud People love to spend their recreational time visiting the Great Barrier Reef World Heritage Area (GBRWHA), (GBRMPA, 2009), and many people are doing it! The recent SELTMP surveys revealed that 95% of residents of coastal town adjacent to the GBR had visited the GBRWHA for recreation at least once, and 87% had visited in the previous 12 months. Many of these visits appeared to be to a mainland beach to walk, swim, and relax. However, 68% of people who told us about their recent trips had been beyond the mainland beach to islands, reefs, shoals, etc., to take part in activities such as fishing, snorkelling and diving. Other activities include boating, sailing, jet skiing, camping, kayaking, sight-seeing, photography, and wildlife viewing, to name a few. Recreational visitors are currently very satisfied with their use of the Marine Park.\ud \ud While most trips beyond the beach were made by ferry, about a third of these trips were accessed by residents' own or someone else's boat. While not everyone is using their vessel very frequently, vessel registration by coastal residents has increased substantially in recent years (Old Department of Transport, unpublished data, 2011).\ud \ud Given all of this activity, it is not surprising that recreation in the GBRWHA provides significant social and cultural benefits as well as many health and wellbeing benefits associated with the psychological interaction with nature (Synergies Economic Consulting, 2012). In economic terms, recreation (defined by Deloitte Access Economics as GBR catchment residents visiting an island, sailing, boating and fishing), contributed $126m in direct value or$243.9m value added to the Australian economy in 2011/12 (Deloitte Access Economics, 2013). This estimate did not include beach visits.\ud Importantly, recreation differs from tourism. The Great Barrier Reef Marine Park Authority define recreation as an independent visit for enjoyment that is not part of a commercial operation (GBRMPA, 2012). For the purposes of the SELTMP Surveys (outline following), any resident of the GBR catchment who visits the GBRWHA is included within recreation; while tourists are defined as those residing outside of the GBR catchment

MightyPix at the LHCb Mighty Tracker — verification of an HV-CMOS pixel chip's digital readout

Abstract MightyPix is a high voltage complementary metal-oxide-semiconductor (HV-CMOS) active pixel sensor, currently being developed for the Mighty Tracker, an upgrade proposed for LHCb in anticipation of the High Luminosity LHC. To ensure that MightyPix will be able to handle the particle hit rates at the Mighty Tracker, which are expected to reach 17 MHz/cm2, simulations of the chip's digital readout mechanism were performed. Using simulated particle hits the chip's performance within the LHCb environment is characterised. For this, a behavioural model of the first prototype, MightyPix1, representing the analogue pixel matrix, together with the synthesised digital logic is used. Simulation results show the MightyPix1 readout mechanism having an efficiency over 99 % up to 20 MHz/cm2. The bottleneck was found to be the speed at which the hits are read out. This yielded new design ideas to improve the readout for MightyPix2, leading to an efficiency of over 99 % up to 30 MHz/cm2.</jats:p

Precursor Ion Independent Algorithm for Top-Down Shotgun Proteomics

We present a precursor ion independent top-down algorithm (PIITA) for use in automated assignment of protein identifications from tandem mass spectra of whole proteins. To acquire the data, we utilize data-dependent acquisition to select protein precursor ions eluting from a C4-based HPLC column for collision induced dissociation in the linear ion trap of an LTQ-Orbitrap mass spectrometer. Gas-phase fractionation is used to increase the number of acquired tandem mass spectra, all of which are recorded in the Orbitrap mass analyzer. To identify proteins, the PIITA algorithm compares deconvoluted, deisotoped, observed tandem mass spectra to all possible theoretical tandem mass spectra for each protein in a genomic sequence database without regard for measured parent ion mass. Only after a protein is identified, is any difference in measured and theoretical precursor mass used to identify and locate post-translation modifications. We demonstrate the application of PIITA to data generated via our wet-lab approach on a Salmonella typhimurium outer membrane extract and compare these results to bottom-up analysis. From these data, we identify 154 proteins by top-down analysis, 73 of which were not identified in a parallel bottom-up analysis. We also identify 201 unique isoforms of these 154 proteins at a false discovery rate (FDR) of <1%

Advances in monitoring the human dimension of natural resource systems: an example from the Great Barrier Reef

The aim of this paper is to demonstrate the feasibility and potential utility of decision-centric social-economic monitoring using data collected from Great Barrier Reef (Reef) region. The social and economic long term monitoring program (SELTMP) for the Reef is a novel attempt to monitor the social and economic dimensions of social-ecological change in a globally and nationally important region. It represents the current status and condition of the major user groups of the Reef with the potential to simultaneously consider trends, interconnections, conflicts, dependencies and vulnerabilities. Our approach was to combine a well-established conceptual framework with a strong governance structure and partnership arrangement that enabled the co-production of knowledge. The framework is a modification of the Millennium Ecosystem Assessment and it was used to guide indicator choice. Indicators were categorised as; (i) resource use and dependency, (ii) ecosystem benefits and well-being, and (iii) drivers of change. Data were collected through secondary datasets where existing and new datasets were created where not, using standard survey techniques. Here we present an overview of baseline results of new survey data from commercial-fishers (n =210), marine-based tourism operators (n =119), tourists (n =2877), local residents (n =3181), and other Australians (n =2002). The indicators chosen describe both social and economic components of the Reef system and represent an unprecedented insight into the ways in which people currently use and depend on the Reef, the benefits that they derive, and how they perceive, value and relate to the Reef and each other. However, the success of a program such as the SELTMP can only occur with well-translated cutting-edge data and knowledge that are collaboratively produced, adaptive, and directly feeds into current management processes. We discuss how data from the SELTMP have already been incorporated into Reef management decision-making through substantial inclusion in three key policy documents

Daptomycin resistance mechanisms in clinically derived Staphylococcus aureus strains assessed by a combined transcriptomics and proteomics approach

Objectives The development of daptomycin resistance in Staphylococcus aureus is associated with clinical treatment failures. The mechanism(s) of such resistance have not been clearly defined. Methods We studied an isogenic daptomycin-susceptible (DAPS) and daptomycin-resistant (DAPR) S. aureus strain pair (616; 701) from a patient with relapsing endocarditis during daptomycin treatment, using comparative transcriptomic and proteomic techniques. Results Minor differences in the genome content were found between strains by DNA hybridization. Transcriptomic analyses identified a number of genes differentially expressed in important functional categories: cell division; metabolism of bacterial envelopes; and global regulation. Of note, the DAPR isolate exhibited reduced expression of the major cell wall autolysis gene coincident with the up-regulation of genes involved in cell wall teichoic acid production. Using quantitative (q)RT-PCR on the gene cadre putatively involved in cationic peptide resistance, we formulated a putative regulatory network compatible with microarray data sets, mainly implicating bacterial envelopes. Of interest, qRT-PCR of this same gene cadre from two distinct isogenic DAPS/DAPR clinical strain pairs revealed evidence of other strain-dependent networks operative in the DAPR phenotype. Comparative proteomics of 616 versus 701 revealed a differential abundance of proteins in various functional categories, including cell wall-associated targets and biofilm formation proteins. Phenotypically, strains 616 and 701 showed major differences in their ability to develop bacterial biofilms in the presence of the antibacterial lipid, oleic acid. Conclusions Compatible with previous in vitro observations, in vivo-acquired DAPR in S. aureus is a complex, multistep phenomenon involving: (i) strain-dependent phenotypes; (ii) transcriptome adaptation; and (iii) modification of the lipid and protein contents of cellular envelope

The Social and Economic Long Term Monitoring Program (SELTMP) 2013: Drivers of change in the Great Barrier Reef

[Extract] Introduction. The Great Barrier Reef region, including the people and industries it supports, is influenced by a range of drivers from global to local scales. We define a driver as any natural or human-induced factor that directly or indirectly causes a change in the GBR system (see SELTMP 2011 for a more in-depth discussion of drivers). Drivers are important to monitor so that we are able: •To understand mechanisms of change in the variables we monitor •To anticipate and begin to predict outcomes •To document the context or "backdrop" of change — in 25 years' time, what will we need to know to interpret change? These drivers themselves change over time (Figure 1) and the direction, magnitude and speed of change can be uncertain; hence monitoring programs need to be adaptive (Lindenmayer and Likens 2009). In this chapter we: 1. Present drivers that were identified in eight SELTMP end user meetings in 2011, the first year of SELTMP, and then present comparative data from a workshop in 2013. 2. We then show how we categorised drivers identified in 2011 and from this, define six driver categories to be monitored. We focus on indirect drivers of change (MEA 2003) — underlying causes of pressures on the GBR —which are most relevant to the social and economic dimensions of the reef. Direct drivers such as biophysical processes of climate change and run-off are monitored by other research programs. However, SELTMP is interested in perceptions of climate change and policies to address it, for example. 3. Present "wishlist" indicators identified by the Drivers of Change Working Group or in the literature for each of the six key categories of drivers, and some of the data collected. Some categories, such as social and cultural drivers, and politics and management, are not easily generalised, and can be highly specific to the GBR region and the different end user groups