Blood plasma has gained protagonism in lipidomics studies due to its availability, uncomplicated collection and preparation, and informative readout of physiological status. At the same time, it is also technically challenging to analyze due to its complex lipid composition affected by many factors, which can hamper the throughput and/or lipidomics coverage. To tackle these issues, we developed a comprehensive, high throughput, and quantitative mass spectrometry-based shotgun lipidomics platform for blood plasma lipid analyses. The main hallmarks of this technology are (i) it is comprehensive, covering 22 quantifiable different lipid classes encompassing more than 200 lipid species; (ii) it is amenable to high-throughput, with less than 5 min acquisition time allowing the complete analysis of 200 plasma samples per day; (iii) it achieves absolute quantification, by inclusion of internal standards for every lipid class measured; (iv) it is highly reproducible, achieving an average coefficient of variation of <10% (intra-day), approx. 10% (inter-day), and approx. 15% (inter-site) for most lipid species; (v) it is easily transferable allowing the direct comparison of data acquired in different sites. Moreover, we thoroughly assessed the influence of blood stabilization with different anticoagulants and freeze-thaw cycles to exclude artifacts generated by sample preparation.Practical applications: This shotgun lipidomics platform can be implemented in different laboratories without compromising reproducibility, allowing multi-site studies and inter-laboratory comparisons. This possibility combined with the high-throughput, broad lipidomic coverage and absolute quantification are important aspects for clinical applications and biomarker research.

Massive water loss is a serious challenge for terrestrial animals, which usually has fatal consequences. However, some organisms have developed means to survive this stress by entering an ametabolic state called anhydrobiosis. The molecular and cellular mechanisms underlying this phenomenon are poorly understood. We recently showed that Caenorhabditis elegans dauer larva, an arrested stage specialized for survival in adverse conditions, is resistant to severe desiccation. However, this requires a preconditioning step at a mild desiccative environment to prepare the organism for harsher desiccation conditions. A systems approach was used to identify factors that are activated during this preconditioning. Using microarray analysis, proteomics, and bioinformatics, genes, proteins, and biochemical pathways that are upregulated during this process were identified. These pathways were validated via reverse genetics by testing the desiccation tolerances of mutants. These data show that the desiccation response is activated by hygrosensation (sensing the desiccative environment) via head neurons. This leads to elimination of reactive oxygen species and xenobiotics, expression of heat shock and intrinsically disordered proteins, polyamine utilization, and induction of fatty acid desaturation pathway. Remarkably, this response is specific and involves a small number of functional pathways, which represent the generic toolkit for anhydrobiosis in plants and animals.

Coordination of multiple kinesin and myosin motors is required for intracellular transport, cell motility and mitosis. However, comprehensive resources that allow systems analysis of the localization and interplay between motors in living cells do not exist. Here, we generated a library of 243 amino- and carboxy-terminally tagged mouse and human bacterial artificial chromosome transgenes to establish 227 stably transfected HeLa cell lines, 15 mouse embryonic stem cell lines and 1 transgenic mouse line. The cells were characterized by expression and localization analyses and further investigated by affinity-purification mass spectrometry, identifying 191 candidate protein-protein interactions. We illustrate the power of this resource in two ways. First, by characterizing a network of interactions that targets CEP170 to centrosomes, and second, by showing that kinesin light-chain heterodimers bind conventional kinesin in cells. Our work provides a set of validated resources and candidate molecular pathways to investigate motor protein function across cell lineages.

Honey bee venom toxins trigger immunological, physiological, and neurological responses within victims. The high occurrence of bee attacks involving potentially fatal toxic and allergic reactions in humans and the prospect of developing novel pharmaceuticals make honey bee venom an attractive target for proteomic studies. Using label-free quantification, we compared the proteome and phosphoproteome of the venom of Africanized honeybees with that of two European subspecies, namely Apis mellifera ligustica and A. m. carnica. From the total of 51 proteins, 42 were common to all three subspecies. Remarkably, the toxins melittin and icarapin were phosphorylated. In all venoms, icarapin was phosphorylated at the (205) Ser residue, which is located in close proximity to its known antigenic site. Melittin, the major toxin of honeybee venoms, was phosphorylated in all venoms at the (10) Thr and (18) Ser residues. (18) Ser phosphorylated melittin-the major of its two phosphorylated forms-was less toxic compared to the native peptide.

Label-free methods streamline quantitative proteomics of tissues by alleviating the need for metabolic labeling of proteins with stable isotopes. Here we detail and implement solutions to common problems in label-free data processing geared toward tissue proteomics by one-dimensional gel electrophoresis followed by liquid chromatography tandem mass spectrometry (geLC MS/MS). Our quantification pipeline showed high levels of performance in terms of duplicate reproducibility, linear dynamic range, and number of proteins identified and quantified. When applied to the liver of an adenomatous polyposis coli (APC) knockout mouse, we demonstrated an 8-fold increase in the number of statistically significant changing proteins compared to alternative approaches, including many more previously unidentified hydrophobic proteins. Better proteome coverage and quantification accuracy revealed molecular details of the perturbed energy metabolism.

No abstract

The formation of water droplets within condensing steam turbines is a complex process that occurs at supersaturated, non-equilibrium conditions and is influenced by the unsteady segmentation of blade wakes by successive blade rows. This is often referred to as ‘wake chopping’, and its effect on the condensation process is the subject of this paper. The practical significance is that thermodynamic ‘wetness losses’ (which constitute a major fraction of the overall loss) are strongly affected by droplet size. Likewise, droplet deposition and the various ensuing two-phase phenomena (such as film migration and coarse-water formation) also depend on the spectrum of droplet sizes in the primary fog. The majority of wake-chopping models presented in the literature adopt a stochastic approach, whereby large numbers of fluid particles are tracked through (some representation of) the turbine flowfield, assigning a random number at each successive blade row to represent the particle’s pitchwise location, and hence its level of dissipation. This study contributes to the existing literature by adding: (a) a comprehensive study of the sensitivity to key model parameters (e.g., blade wake shape and wake decay rate); (b) an assessment of the impact of circumferential pressure variations; (c) a study of the implications for wetness losses and (d) a study of the implications for deposition rates.

Surface tension, [Jm −2 ] τ Wake decay time, [s] τ p Particle relaxation time, [s] φ Kantrowitz correction factor ψ Mass flow function Ω Rotational speed, [s −1 ] Subscripts * Critical value l Liquid phase ls Last stage g Gas phase m Mixture r Radial direction s Saturation value 32 Sauter mean value θ Circumferential direction ∞ Average value