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

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

Characterization of colon cancer cells: a functional approach characterizing CD133 as a potential stem cell marker

<p>Abstract</p> <p>Background</p> <p>Isolation and characterization of tumourigenic colon cancer initiating cells may help to develop novel diagnostic and therapeutic procedures.</p> <p>Methods</p> <p>We characterized a panel of fourteen human colon carcinoma cell lines and their corresponding xenografts for the surface expression of potential stem cell markers CD133, CD24, CD44, CDCP1 and CXCR4. In five cell lines and nine xenografts, mRNA expression of these markers was determined. Tumour growth behaviour of CD133+, CD133- and unsorted SW620 cells was evaluated <it>in vivo</it>.</p> <p>Results</p> <p>All five putative stem cell markers showed distinct expression patterns in the tumours examined. Two patient-derived cell lines highly expressed CD133 (> 85% of positive cells) and three other cell lines had an expression level of about 50% whereas in long-term culture based models CD133 expression ranged only from 0 to 20%. In 8/14 cell lines, more than 80% of the cells were positive for CD24 and 11/14 were over 70% positive for CD44. 10/14 cell lines expressed CDCP1 on ≥ 83% of cells. CXCR4 expression was determined solely on 94 L and SW480.</p> <p>Analyses of the corresponding xenografts revealed a significant reduction of cell numbers expressing the investigated surface markers and showed single cell fractions expressing up to three markers simultaneously.</p> <p>Statistical analysis revealed that the CXCR4 mRNA level correlates negatively with the protein expression of CD133, CD44, CD24 and CDCP1 in cell lines and xenografts.</p> <p>A lower differentiation grade of donor material correlated with a higher CDCP1 mRNA expression level in the respective tumour model.</p> <p><it>In vivo </it>growth behaviour studies of SW620 revealed significantly higher take rates and shorter doubling times in the tumour growth of CD133 positive subclones in comparison to the unsorted cell line or CD133 negative subclones.</p> <p>Conclusions</p> <p>Our data revealed correlations in the expression of surface markers CD44 and CD24 as well as CD44 and CDCP1 and strongly suggest that CD133 is a stem cell marker within our colon carcinoma panel. Further studies will elucidate its role as a potential therapeutic target.</p