Generation of accurate peptide retention data for targeted and data independent quantitative LC‐MS analysis: Chromatographic lessons in proteomics

The emergence of data‐independent quantitative LC‐MS/MS analysis protocols further highlights the importance of high‐quality reproducible chromatographic procedures. Knowing, controlling and being able to predict the effect of multiple factors that alter peptide RP‐HPLC separation selectivity is critical for successful data collection for the construction of ion libraries. Proteomic researchers have often regarded RP‐HPLC as a “black box”, while vast amount of research on peptide separation is readily available. In addition to obvious parameters, such as the type of ion‐pairing modifier, stationary phase and column temperature, we describe the “mysterious” effects of gradient slope, column size and flow rate on peptide separation selectivity. Retention time variations due to these parameters are governed by the linear solvent strength (LSS) theory on a peptide level by the value of its slope S in the basic LSS equation—a parameter that can be accurately predicted. Thus, the application of shallower gradients, higher flow rates, or smaller columns will each increases the relative retention of peptides with higher S‐values (long species with multiple positively charged groups). Simultaneous changes to these parameters that each drive shifts in separation selectivity in the same direction should be avoided. The unification of terminology represents another pressing issue in this field of applied proteomics that should be addressed to facilitate further progress.     

Description of a cryptic thermophilic (pro)phage,CBP1 from Caldibacillus debilis strain GB1

Extremophiles - This study characterizes a cryptic (pro)phage-related sequence within the Caldibacillus debilis GB1 genome, designated CBP1.CBP1 is a Siphoviridae-like genome highly related to...     

Changes in fatty acids contents and growth characteristics in transformed oilseed rape (Brassica napus)

Spring oilseed rapeBrassica napus L. ssp.oleifera cv. HM-81 was transformed with T_L-DNA of the Ri plasmid of the agropine strainAgrobacterium rhizogenes 15834. Selfed progenies (R₂ and R₃ generations) were studied for changes in values of growth characteristics and fatty acids contents. Transformants are ‘homozygous’ for T_L-DNA. Both generations of transformants differed significantly from the nontransformed control plants in reduced length, lower number of pods per plant, lower total mass of seeds and the higher number of branches. The contents of palmitic, linoleic and linolenic acids were significantly higher in transformants when compared with the control. On the contrary, the contents of both stearic and oleic acids were in most of transformants significantly lower. Only traces of erucic acid (less than 0.05 % ) were found, both in transformed and nontransformed plants.     

Diversity and ecology of algae from the Nahal Qishon river,northern Israel

In 59 samples of periphyton and phytoplankton collected in 2002 - 2003 from the Nahal Qishon (Qishon River), northern Israel, we found 178 species from seven divisions of algae and cyanoprocaryotes. Diatoms, clorophytes, and cyanoprocaryotes prevail. Nitzschia and Navicula (Bacillariophyta) are the most abundant. Most of the species are cosmopolitan or widespread, except Lagynion janei (Chrysophyta), which is endemic for the Mediterranean Realm. About 17% of species (26) are new for Israel and five of them represent the first recorded genera: Crinalium endophyticum Crow, Actinocyclus normanii (Gregory) Hustedt, Rhizoclonium hieroglyphicum (Agardh) Kütz (Chlorophyta), Lagynion janei Bourelly, and Stylococcus aureus Chodat. Most of them come from a rare riverine assemblage with red alga Audouinella pygmea, as well as from the estuarine assemblage. Alkaliphiles predominate among the indicators of acidity, with few acidophiles confined to the communities under the impact of industrial wastes. Among the indicators of salinity, most numerous are the oligohalobien-indifferents and species adapted to a moderate salinity level. The relative species richness of ecological groups and the indices of saprobity are correlated with changes in conductivity, pH, and N-nitrate concentration. Indicators of organic pollution fall in the range of betameso- to alfamesosaprobic self-purification grades. Our studies show ecological significance of the Nahal Qishon as a model for a strongly disturbed aquatic ecosystem in the coastal zone of eastern Mediterranean.     

Influence of the radiation of the plasma-focus current sheath on the implosion dynamics of condensed targets

Results are presented from experimental and theoretical studies of the influence of the radiation of the plasma-focus current sheath on the implosion dynamics of condensed targets. Radiative losses from the current sheath of a plasma focus in neon, argon, and hydrogen with a 2% admixture of Xe are calculated with allowance for the line, bremsstrahlung, and recombination radiation. It is shown that the temperature of the neon plasma (10–15 eV) is quite sufficient to evaporate Al₂O₃ grains of radii 10–20 μm. The use of neon as a working gas makes it possible to alter the cold-start condition in experiments on the implosion of foam liners.     

Citrus chlorophyllase dynamics at ethylene-induced fruit color-break: a study of chlorophyllase expression, posttranslational processing kinetics, and in situ intracellular localization

Fruit color-break is the visual manifestation of the developmentally regulated transition of chloroplasts to chromoplasts during fruit ripening and often involves biosynthesis of copious amounts of carotenoids concomitant with massive breakdown of chlorophyll. Regulation of chlorophyll breakdown at different physiological and developmental stages of the plant life cycle, particularly at fruit color-break, is still not well understood. Here, we present the dynamics of native chlorophyllase (Chlase) and chlorophyll breakdown in lemon (Citrus limon) fruit during ethylene-induced color-break. We show, using in situ immunofluorescence on ethylene-treated fruit peel (flavedo) tissue, that citrus Chlase is located in the plastid, in contrast to recent reports suggesting cytoplasmic localization of Arabidopsis (Arabidopsis thaliana) Chlases. At the intra-organellar level, Chlase signal was found to overlap mostly with chlorophyll fluorescence, suggesting association of most of the Chlase protein with the photosynthetic membranes. Confocal microscopy analysis showed that the kinetics of chlorophyll breakdown was not uniform in the flavedo cells. Chlorophyll quantity at the cellular level was negatively correlated with plastid Chlase accumulation; plastids with reduced chlorophyll content were found by in situ immunofluorescence to contain significant levels of Chlase, while plastids containing still-intact chlorophyll lacked any Chlase signal. Immunoblot and protein-mass spectrometry analyses were used to demonstrate that citrus Chlase initially accumulates as an approximately 35-kD precursor, which is subsequently N-terminally processed to approximately 33-kD mature forms by cleavage at either of three consecutive amino acid positions. Chlase plastid localization, expression kinetics, and the negative correlation with chlorophyll levels support the central role of the enzyme in chlorophyll breakdown during citrus fruit color-break.     

Concentrations and ecological risks of metals in surface sediments of some coastal creeks in the Niger Delta,Nigeria

The concentrations of nine metals were measured by atomic absorption spectrophotometry in surface sediments of three coastal creeks, namely, the Ifie, Egbokodo and Ubeji creeks, in the Niger Delta of Nigeria, from August 2012 to January 2013. The aim of the study was to provide information on the spatial and seasonal distribution patterns, degree of contamination, and ecological risks of metals in these sediments. The mean concentrations of the nine metals in these creek sediments ranged from 0.30 to 3.20?mg kg^?1 Cd; 10.7 to 24.7?mg kg^?1 Pb, 125 to 466?mg kg^?1 Cr; 3.1.10 to 14.9?mg kg^?1 Cu; 4.7 to 14.3?mg kg^?1 Co; 61.1 to 115?mg kg^?1 Ni; 106 to 183?mg kg^?1 Mn; 52.0 to 170?mg kg^?1 Zn and 5 469 to 20 639?mg kg^?1 Fe. In general, the metal concentrations were higher in the dry season than the wet season, except for Cr. The concentrations of Cd, Cr, Ni and Zn were above their regulatory control limits in sediment as specified by the Nigerian Regulatory Authority and Cd was identified as the main ecological risk factor. The enrichment factors for the studied metals followed the order: Cd > Cr > Ni > Zn > Pb > Co > Mn > Cu. The average multiple pollution index values indicated that these sediments were severely polluted with significant inputs from Cd, Ni and Cr.     

Proteomic characterization of serine hydrolase activity and composition in normal urine

Background

Serine hydrolases constitute a large enzyme family involved in a diversity of proteolytic and metabolic processes which are essential for many aspects of normal physiology. The roles of serine hydrolases in renal function are largely unknown and monitoring their activity may provide important insights into renal physiology. The goal of this study was to profile urinary serine hydrolases with activity-based protein profiling (ABPP) and to perform an in-depth compositional analysis.

Methods

Eighteen healthy individuals provided random, mid-stream urine samples. ABPP was performed by reacting urines (n = 18) with a rhodamine-tagged fluorophosphonate probe and visualizing on SDS-PAGE. Active serine hydrolases were isolated with affinity purification and identified on MS-MS. Enzyme activity was confirmed with substrate specific assays. A complementary 2D LC/MS-MS analysis was performed to evaluate the composition of serine hydrolases in urine.

Results

Enzyme activity was closely, but not exclusively, correlated with protein quantity. Affinity purification and MS/MS identified 13 active serine hydrolases. The epithelial sodium channel (ENaC) and calcium channel (TRPV5) regulators, tissue kallikrein and plasmin were identified in active forms, suggesting a potential role in regulating sodium and calcium reabsorption in a healthy human model. Complement C1r subcomponent-like protein, mannan binding lectin serine protease 2 and myeloblastin (proteinase 3) were also identified in active forms. The in-depth compositional analysis identified 62 serine hydrolases in urine independent of activity state.

Conclusions

This study identified luminal regulators of electrolyte homeostasis in an active state in the urine, which suggests tissue kallikrein and plasmin may be functionally relevant in healthy individuals. Additional serine hydrolases were identified in an active form that may contribute to regulating innate immunity of the urinary tract. Finally, the optimized ABPP technique in urine demonstrates its feasibility, reproducibility and potential applicability to profiling urinary enzyme activity in different renal physiological and pathophysiological conditions.