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Enrichment Techniques (enrichment + techniques)
Selected AbstractsTechniques for phosphopeptide enrichment prior to analysis by mass spectrometryMASS SPECTROMETRY REVIEWS, Issue 1 2010Jamie D. Dunn Abstract Mass spectrometry is the tool of choice to investigate protein phosphorylation, which plays a vital role in cell regulation and diseases such as cancer. However, low abundances of phosphopeptides and low degrees of phosphorylation typically necessitate isolation and concentration of phosphopeptides prior to MS analysis. This review discusses the enrichment of phosphopeptides with immobilized metal affinity chromatography, reversible covalent binding, and metal oxide affinity chromatography. Capture of phosphopeptides on TiO2 seems especially promising in terms of selectivity and recovery, but the success of all methods depends on careful selection of binding, washing, and elution solutions. Enrichment techniques are complementary, such that a combination of methods greatly enhances the number of phosphopeptides isolated from complex samples. Development of a standard series of phosphopeptides in a highly complex mixture of digested proteins would greatly aid the comparison of different enrichment methods. Phosphopeptide binding to magnetic beads and on-plate isolation prior to MALDI-MS are emerging as convenient methods for purification of small (µL) samples. On-plate enrichment can yield >70% recoveries of phosphopeptides in mixtures of a few digested proteins and can avoid sample-handling steps, but this technique is likely limited to relatively simple samples such as immunoprecipitates. With recent advances in enrichment techniques in hand, MS analysis should provide important insights into phosphorylation pathways. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:29,54, 2010 [source] Rhodotorula cycloclastica sp. nov., Rhodotorula retinophila sp. nov., and Rhodotorula terpenoidalis sp. nov., three limonene-utilizing yeasts isolated from soilFEMS YEAST RESEARCH, Issue 8 2004Vu Nguyen Thanh Abstract During a search for yeasts that hydroxylate monoterpenes, four yeast strains were isolated from soil and plant residue in monoterpene-rich environments using enrichment techniques with cyclohexanedioic acid or cyclohexanedimethanol as sole carbon source. These strains were able to utilize (+)-limonene supplied as a vapor as only carbon source. The yeasts have a CoQ-10 system. Morphology and physiological properties of the strains did not fit any known yeast species. Recent analysis of the 26S D1/D2 and ITS-5.8S rDNA sequences of basidiomycetous yeasts showed that these strains represented three hitherto unknown species of Rhodotorula and fell in a cluster consisting of Rhodotorula philyla and the mycoparasitic fungus Colacogloea peniophorae. Descriptions of three new species Rhodotorula cycloclastica (type strain TVN 309=UOFS Y 2046=CBS 8448), Rhodotorula retinophila (type strain TVN 295=UOFS Y 2043=CBS 8446), Rhodotorula terpenoidalis (type strain TVN 310=UOFS Y 2042=CBS 8445) are proposed to accommodate these isolates. [source] Partition of unity enrichment for bimaterial interface cracksINTERNATIONAL JOURNAL FOR NUMERICAL METHODS IN ENGINEERING, Issue 8 2004N. Sukumar Abstract Partition of unity enrichment techniques are developed for bimaterial interface cracks. A discontinuous function and the two-dimensional near-tip asymptotic displacement functions are added to the finite element approximation using the framework of partition of unity. This enables the domain to be modelled by finite elements without explicitly meshing the crack surfaces. The crack-tip enrichment functions are chosen as those that span the asymptotic displacement fields for an interfacial crack. The concept of partition of unity facilitates the incorporation of the oscillatory nature of the singularity within a conforming finite element approximation. The mixed-mode (complex) stress intensity factors for bimaterial interfacial cracks are numerically evaluated using the domain form of the interaction integral. Good agreement between the numerical results and the reference solutions for benchmark interfacial crack problems is realized. Copyright © 2004 John Wiley & Sons, Ltd. [source] Distinct properties of murine ,5 ,-aminobutyric acid type a receptors revealed by biochemical fractionation and mass spectroscopyJOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2009Young H. Ju Abstract ,-Aminobutyric acid type A receptors (GABAARs) that contain the ,5 subunit are expressed predominantly in the hippocampus, where they regulate learning and memory processes. Unlike conventional postsynaptic receptors, GABAARs containing the ,5 subunit (,5 GABAARs) are localized primarily to extrasynaptic regions of neurons, where they generate a tonic inhibitory conductance. The unique characteristics of ,5 GABAARs have been examined with pharmacological, immunostaining, and electrophysiological techniques; however, little is known about their biochemical properties. The aim of this study was to modify existing purification and enrichment techniques to isolate ,5 GABAARs preferentially from the mouse hippocampus and to identify the ,5 subunit by using tandem mass spectroscopy (MS/MS). The results showed that the detergent solubility of the ,5 subunits was distinct from that of ,1 and ,2 subunits, and the relative distribution of the ,5 subunits in Triton X-100-soluble fractions was correlated with that of the extracellular protein radixin but not with that of the postsynaptic protein gephyrin. Mass spectrometry identified the ,5 subunit and showed that this subunit associates with multiple ,, ,, and , subunits, but most frequently the ,3 subunit. Thus, the ,5 subunits coassemble with similar subunits as their synaptic counterparts yet have a distinct detergent solubility profile. Mass spectroscopy now offers a method for detecting and characterizing factors that confer the unique detergent solubility and possibly cellular location of ,5 GABAARs in hippocampal neurons. © 2009 Wiley-Liss, Inc. [source] Techniques for phosphopeptide enrichment prior to analysis by mass spectrometryMASS SPECTROMETRY REVIEWS, Issue 1 2010Jamie D. Dunn Abstract Mass spectrometry is the tool of choice to investigate protein phosphorylation, which plays a vital role in cell regulation and diseases such as cancer. However, low abundances of phosphopeptides and low degrees of phosphorylation typically necessitate isolation and concentration of phosphopeptides prior to MS analysis. This review discusses the enrichment of phosphopeptides with immobilized metal affinity chromatography, reversible covalent binding, and metal oxide affinity chromatography. Capture of phosphopeptides on TiO2 seems especially promising in terms of selectivity and recovery, but the success of all methods depends on careful selection of binding, washing, and elution solutions. Enrichment techniques are complementary, such that a combination of methods greatly enhances the number of phosphopeptides isolated from complex samples. Development of a standard series of phosphopeptides in a highly complex mixture of digested proteins would greatly aid the comparison of different enrichment methods. Phosphopeptide binding to magnetic beads and on-plate isolation prior to MALDI-MS are emerging as convenient methods for purification of small (µL) samples. On-plate enrichment can yield >70% recoveries of phosphopeptides in mixtures of a few digested proteins and can avoid sample-handling steps, but this technique is likely limited to relatively simple samples such as immunoprecipitates. With recent advances in enrichment techniques in hand, MS analysis should provide important insights into phosphorylation pathways. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:29,54, 2010 [source] Characterization of microsatellite loci in the endangered species of fern Marsilea strigosa Willd. (Marsileaceae, Pteridophyta)MOLECULAR ECOLOGY RESOURCES, Issue 1-2 2001R. Vitalis Abstract Absence of enzymatic polymorphism in the endangered species of fern Marsilea strigosa prompted us to develop microsatellite loci in this species. We used an enriched partial genomic library, from which six polymorphic microsatellite loci were obtained. From a previous attempt to develop microsatellite markers with standard techniques, a single long and polymorphic locus was obtained. This highlights the benefits from using enrichment techniques in those species where microsatellite loci may be rare in the genome. [source] A new approach to determine method detection limits for compound-specific isotope analysis of volatile organic compoundsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 24 2006Maik A. Jochmann Compound-specific isotope analysis (CSIA) has been established as a useful tool in the field of environmental science, in particular in the assessment of contaminated sites. What limits the use of gas chromatography/isotope ratio mass spectrometry (GC/IRMS) is the low sensitivity of the method compared with GC/MS analysis; however, the development of suitable extraction and enrichment techniques for important groundwater contaminants will extend the fields of application for GC/IRMS. So far, purge and trap (P&T) is the most effective, known preconcentration technique for on-line CSIA with the lowest reported method detection limits (MDLs in the low,µg/L range). With the goal of improving the sensitivity of a fully automated GC/IRMS analysis method, a commercially available P&T system was modified. The method was evaluated for ten monoaromatic compounds (benzene, toluene, para -xylene, ethylbenzene, propylbenzene, isopropylbenzene, 1,2,3-trimethylbenzene, 1,2,4-trimethylbenzene, 1,3,5-trimethylbenzene, fluorobenzene) and ten halogenated volatile organic compounds (VOCs) (dichloromethane, cis -1,2-dichloroethene, trans -1,2-dichloroethene, carbon tetrachloride, chloroform, 1,2-dichloroethane, trichloroethene, tetrachlorethene, 1,2-dibromoethane, bromoform). The influence of method parameters, including purge gas flow rates and purge times, on ,13C values of target compounds was evaluated. The P&T method showed good reproducibility, high linearity and small isotopic fractionation. MDLs were determined by consecutive calculation of the ,13C mean values. The last concentration for which the ,13C value was within this iterative interval and for which the standard deviation was lower than ±0.5, for triplicate measurements was defined as the MDL. MDLs for monoaromatic compounds between 0.07 and 0.35,µg/L are the lowest values reported so far for continuous-flow isotope ratio measurements using an automated system. MDLs for halogenated hydrocarbons were between 0.76 and 27,µg/L. The environmental applicability of the P&T-GC/IRMS method in the low-µg/L range was demonstrated in a case study on groundwater samples from a former military air field contaminated with VOCs. Copyright © 2006 John Wiley & Sons, Ltd. [source] Improved techniques for rearing mud crab Scylla paramamosain (Estampador 1949) larvaeAQUACULTURE RESEARCH, Issue 14 2007Truong Trong Nghia Abstract A series of rearing trials in small 1 L cones and large tanks of 30,100 L were carried out to develop optimal rearing techniques for mud crab (Scylla paramamosain) larvae. Using water exchange (discontinuous partial water renewal or continuous treatment through biofiltration) and micro-algae (Chlorella or Chaetoceros) supplementation (daily supplementation at 0.1,0.2 million cells mL,1 or maintenance at 1,2 millions cells mL,1), six different types of rearing systems were tried. The combination of a green-water batch system for early stages and a recirculating system with micro-algae supplementation for later stages resulted in the best overall performance of the crab larvae. No clear effects of crab stocking density (50,200 larvae L,1) and rotifer (30,60 rotifers mL,1) and Artemia density (10,20 L,1) were observed. A stocking density of 100,150 zoea 1 (Z1) L,1, combined with rotifer of 30,45 mL,1 for early stages and Artemia feeding at 10,15 nauplii mL,1 for Z3,Z5 seemed to produce the best performance of S. paramamosain larvae. Optimal rations for crab larvae should, however, be adjusted depending on the species, larval stage, larval status, prey size, rearing system and techniques. A practical feeding schedule could be to increase live food density from 30 to 45 rotifers mL,1 from Z1 to Z2 and increase the number of Artemia nauplii mL,1 from 10 to 15 from Z3 to Z5. Bacterial disease remains one of the key factors underlying the high mortality in the zoea stages. Further research to develop safe prophylactic treatments is therefore warranted. Combined with proper live food enrichment techniques, application of these findings has sustained a survival rate from Z1 to crab 1,2 stages in large rearing tanks of 10,15% (maximum 30%). [source] | |||||||||||||