Skyline is a freely available, open-source Windows client application for accelerating targeted proteomics experimentation, with an emphasis on the proteomics and mass spectrometry community as users and as contributors. This review covers the informatics encompassed by the Skyline ecosystem, from computationally assisted targeted mass spectrometry method development, to raw acquisition file data processing, and quantitative analysis and results sharing.

Bacteria and Archaea play critical roles in marine energy fluxes and nutrient cycles by incorporating and redistributing dissolved organic matter and inorganic nutrients in the oceans. How these microorganisms do this work at the level of the expressed protein is known only from a few studies of targeted lineages. We used comparative membrane metaproteomics to identify functional responses of communities to different nutrient concentrations on an oceanic scale. Comparative analyses of microbial membrane fractions revealed shifts in nutrient utilization and energy transduction along an environmental gradient in South Atlantic surface waters, from a low-nutrient gyre to a highly productive coastal upwelling region. The dominant membrane proteins identified (19%) were TonB-dependent transporters (TBDTs), which are known to utilize a proton motive force to transport nutrients across the outer membrane of Gram-negative bacteria. The ocean-wide importance of TonB-dependent nutrient acquisition in marine bacteria was unsuspected. Diverse light-harvesting rhodopsins were detected in membrane proteomes from every sample. Proteomic evidence of both TBDTs and rhodopsins in the same lineages suggest that phototrophic bacterioplankton have the potential to use energy from light to fuel transport activities. We also identified viral proteins in every sample and archaeal ammonia monooxygenase proteins in the upwelling region, suggesting that Archaea are important nitrifiers in nutrient-rich surface waters.

A changing climate is altering many ocean properties that consequently will modify marine productivity. Previous phytoplankton manipulation studies have focused on individual or subsets of these properties. Here, we investigate the cumulative e ects of multi-faceted change on a subantarctic diatom Pseudonitzschia multiseries by concurrently manipulating five stressors (light/nutrients/CO 2 /temperature/iron) that primarily control its physiology, and explore underlying reasons for altered physiological performance. Climate change enhances diatom growth mainly owing to warming and iron enrichment, and both properties decrease cellular nutrient quotas, partially o setting any e ects of decreased nutrient supply by 2100. Physiological diagnostics and comparative proteomics demonstrate the joint importance of individual and interactive e ects of temperature and iron, and reveal biased future predictions from experimental outcomes when only a subset of multi-stressors is considered. Our findings for subantarctic waters illustrate how composite regional studies are needed to provide accurate global projections of future shifts in productivity and distinguish underlying species-specific physiological mechanisms.A n ongoing major challenge is to grasp how climate-changemediated alteration of environmental conditions will influence biota across different scales, from organismal health to community structure 1,2 . Oceanographers have employed climate-change models 3,4 , time-series observations 5 and manipulation experiments 6 to understand the biological ramifications of global change. Phytoplankton manipulation studies reveal how alteration of individual properties, such as CO 2 , affects physiology 2,6,7 . However, the validity of such singleparameter findings 6,8,9 , in the context of complex ocean change 1,2 , is challenged by research that reveals interactive effects between multi-stressors on phytoplankton physiology 10,11 . We need to diagnose and understand the physiological mechanisms that underpin interconnected responses to multi-stressors, which together set the cumulative response of phytoplankton species to changing conditions 4,6,8 .Understanding the combined effects, across the global ocean, of complex change on phytoplankton physiology requires a gradualist approach 12,13 . Individual provinces will encounter different permutations of multi-faceted change 14 , and each is characterized by a range of resident phytoplankton groups (termed biomes 5 ). Earth System models provide a framework of projections of regional change 14 that stimulate improved experimental design to understand the biological effects of oceanic change. In return, a new generation of manipulation studies must deliver estimates of the combined effects of complex change on many phytoplankton species, and distinguish the underlying mechanisms that underpin these physiological outcomes.Here, we target subantarctic diatoms, which are ubiquitous and bloom-formers 15 . We experimentally manipulate a representative species 6,15 (Pseudonitzschi...

Phytoplankton growth rates are limited by the supply of iron (Fe) in approximately one third of the open ocean, with major implications for carbon dioxide sequestration and carbon (C) biogeochemistry. To date, understanding how alteration of Fe supply changes phytoplankton physiology has focused on traditional metrics such as growth rate, elemental composition, and biophysical measurements such as photosynthetic competence (Fv/Fm). Researchers have subsequently employed transcriptomics to probe relationships between changes in Fe supply and phytoplankton physiology. Recently, studies have investigated longer-term (i.e. following acclimation) responses of phytoplankton to various Fe conditions. In the present study, the coastal diatom, Thalassiosira pseudonana, was acclimated (10 generations) to either low or high Fe conditions, i.e. Fe-limiting and Fe-replete. Quantitative proteomics and a newly developed proteomic profiling technique that identifies low abundance proteins were employed to examine the full complement of expressed proteins and consequently the metabolic pathways utilized by the diatom under the two Fe conditions. A total of 1850 proteins were confidently identified, nearly tripling previous identifications made from differential expression in diatoms. Given sufficient time to acclimate to Fe limitation, T. pseudonana up-regulates proteins involved in pathways associated with intracellular protein recycling, thereby decreasing dependence on extracellular nitrogen (N), C and Fe. The relative increase in the abundance of photorespiration and pentose phosphate pathway proteins reveal novel metabolic shifts, which create substrates that could support other well-established physiological responses, such as heavily silicified frustules observed for Fe-limited diatoms. Here, we discovered that proteins and hence pathways observed to be down-regulated in short-term Fe starvation studies are constitutively expressed when T. pseudonana is acclimated (i.e., nitrate and nitrite transporters, Photosystem II and Photosystem I complexes). Acclimation of the diatom to the desired Fe conditions and the comprehensive proteomic approach provides a more robust interpretation of this dynamic proteome than previous studies.

Significance Microscopic marine algae (phytoplankton) are responsible for much of Earth's photosynthesis, serving as the base of a massive food web supporting fisheries. Phytoplankton compete for limiting resources, with some species producing noxious compounds that kill competitors or inhibit their growth. The red-tide dinoflagellate Karenia brevis is one such allelopathic species, causing growth suppression of other phytoplankton and negatively impacting coastal ecosystems. Metabolomic and proteomic approaches were used to characterize the sublethal physiological impacts of K. brevis allelopathy on two competing phytoplankton, providing insights into the physiological mechanisms by which allelopathy occurs and the metabolic pathways that enable resistance in co-occurring competitors.

Bacterial ADP-ribosyltransferases (ADPRTs) are toxins that play a significant role in pathogenicity by inactivating host proteins through covalent addition of ADP-ribose. In this study we used ADP-ribosylated Kemptide as a standard to examine the effectiveness of three common tandem mass spectrometry fragmentation methods for assignment of amino acid sequence and site of modification. Fragmentation mechanisms investigated include low-energy collision induced dissociation (CID), infrared multiphoton dissociation (IRMPD), and electron capture dissociation (ECD); all were performed on a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer. We show that ECD, but neither CID nor IRMPD, of ADP-ribosylated-Kemptide produces tandem mass spectra that are interpretable with regard to amino acid sequence assignment and site of modification. Examination of CID and IRMPD tandem mass spectra of ADP-ribosylated Kemptide revealed that fragmentation was primarily focused to the ADP-ribose region, generating several potential diagnostic ions for use in discovery of ADP-ribosylated proteins. Due to the lower relative sensitivity of ECD during data-dependent acquisition to CID, we suggest a two-fold strategy where CID and IRMPD are first used to detect ADP-ribosylated peptides, followed by sequence assignment and location of modification by ECD analysis.

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BackgroundOcean acidification as a result of increased anthropogenic CO2 emissions is occurring in marine and estuarine environments worldwide. The coastal ocean experiences additional daily and seasonal fluctuations in pH that can be lower than projected end-of-century open ocean pH reductions. In order to assess the impact of ocean acidification on marine invertebrates, Pacific oysters (Crassostrea gigas) were exposed to one of four different p CO2 levels for four weeks: 400 μatm (pH 8.0), 800 μatm (pH 7.7), 1000 μatm (pH 7.6), or 2800 μatm (pH 7.3).ResultsAt the end of the four week exposure period, oysters in all four p CO2 environments deposited new shell, but growth rate was not different among the treatments. However, micromechanical properties of the new shell were compromised by elevated p CO2. Elevated p CO2 affected neither whole body fatty acid composition, nor glycogen content, nor mortality rate associated with acute heat shock. Shotgun proteomics revealed that several physiological pathways were significantly affected by ocean acidification, including antioxidant response, carbohydrate metabolism, and transcription and translation. Additionally, the proteomic response to a second stress differed with p CO2, with numerous processes significantly affected by mechanical stimulation at high versus low p CO2 (all proteomics data are available in the ProteomeXchange under the identifier PXD000835).ConclusionsOyster physiology is significantly altered by exposure to elevated p CO2, indicating changes in energy resource use. This is especially apparent in the assessment of the effects of p CO2 on the proteomic response to a second stress. The altered stress response illustrates that ocean acidification may impact how oysters respond to other changes in their environment. These data contribute to an integrative view of the effects of ocean acidification on oysters as well as physiological trade-offs during environmental stress.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-951) contains supplementary material, which is available to authorized users.