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Isolation of microgram amounts of proteins and submicrogram quantities of peptides in a form suitable for sequence analysis is a key step in high sensitivity protein sequencing technology. Recently, methods have been developed which allow the direct, high yield, recovery of microgram amounts of sequenceable proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis. In the present publication, we describe an extension of these methods to obtain N-terminal or internal sequence information from proteins separated by isoelectric focusing in polyacrylamide gels containing immobilized pH gradients. For N-terminal sequence analysis, separated proteins were electrophoretically transferred (electroblotted) onto chemically-modified glass fiber sheets, a support compatible with Edman degradation chemistry. Transferred protein bands were detected on the support, cut out and directly inserted into the cartridge of a gas-phase protein sequenator. For internal sequence analysis, separated proteins were electrophoretically transferred onto nitrocellulose. Protein bands were detected by staining, cut out and the proteins subjected to enzymatic digestion directly on the support. The resulting cleavage fragments (peptides) were released into the supernatant where they were recovered and separated by narrow-bore reversed-phase high performance liquid chromatography for sequence analysis. The potential of this methodology is illustrated by the comparative peptide mapping of isoforms of bovine carbonic anhydrase. 相似文献
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Hood BL Lucas DA Kim G Chan KC Blonder J Issaq HJ Veenstra TD Conrads TP Pollet I Karsan A 《Journal of the American Society for Mass Spectrometry》2005,16(8):1221-1230
With advancements in the analytical technologies and methodologies in proteomics, there is great interest in biomarker discovery in biofluids such as serum and plasma. Current hypotheses suggest that the low molecular weight (LMW) serum proteome possesses an archive of clipped and cleaved protein fragments that may provide insight into disease development. Though these biofluids represent attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. Mice are one of the most extensively used animal models for studying human disease because they represent a highly controllable experimental model system. In this study, the LMW serum proteome was compared between xenografted tumor-bearing mice and control mice by differential labeling utilizing trypsin-mediated incorporation of the stable isotope of oxygen, 18O. The digestates were combined, fractionated by strong cation exchange chromatography, and analyzed by nanoflow reversed-phase liquid chromatography coupled online with tandem mass spectrometry, resulting in the identification of 6003 proteins identified by at least a single, fully tryptic peptide. Almost 1650 proteins were identified and quantitated by two or more fully tryptic peptides. The methodology adopted in this work provides the means for future quantitative measurements in comparative animal models of disease and in human disease cohorts. 相似文献
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Pulsed field gel electrophoresis: studies of DNA migration made with the programmable, autonomously-controlled electrode electrophoresis system 总被引:2,自引:0,他引:2
We have studied the migration of DNA in pulsed field agarose gels under a variety of electrophoresis conditions. We have made use of an instrument which can generate electric fields of any orientation, magnitude, or duration to compare different separation techniques for DNA molecules of from 1 to several thousand kilobase pairs. We discuss the capabilities of the system and present results of gel runs in which electrophoresis conditions were changed individually or in combination. The mobility of DNA in pulsed field gels is shown to reflect a number of interdependent physical parameters. 相似文献
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Garcia Ruano JL Alemparte C Martin Castro AM Adams H Rodriguez Ramos JH 《The Journal of organic chemistry》2000,65(23):7938-7943
The behavior of (Z)-3-p-tolylsulfinylacrylonitrile (1) as a chiral dienophile has been evaluated from its reactions with furan and acyclic dienes. Electrostatic interactions of the cyano group with the sulfinyl one restrict the conformational mobility around the C-S bond, thus controlling the pi-facial selectivity, which is almost complete in all cases, the approach of the diene from the less-hindered face of the dienophile (that bearing the lone electron pair) in the predominant rotamer being the favored one. The regioselectivity is also completely controlled by the cyano group. Additionally, the reactivity of compound 1 as well as its endo-selectivity are both higher than those observed for the corresponding (Z)-3-sulfinylacrylates, thus proving the potential of sulfinylnitriles as chiral dienophiles. 相似文献
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EI Rodriguez Martinez CI Barriga-Paulino MI Zapata CC Chinchilla AM López-Jiménez CM Gómez 《BMC neuroscience》2012,13(1):104-23
ABSTRACT: BACKGROUND: The peri-adolescent period is a crucial developmental moment of transition from childhood to emergent adulthood. The present report analyses the differences in Power Spectrum (PS) of the Electroencephalogram (EEG) between late childhood (24 children between 8 and 13 years old) and young adulthood (24 young adults between 18 and 23 years old). RESULTS: The narrow band analysis of the Electroencephalogram was computed in the frequency range of 0--20 Hz. The analysis of mean and variance suggested that six frequency ranges presented a different rate of maturation at these ages, namely: low delta, delta-theta, low alpha, high alpha, low beta and high beta. For most of these bands the maturation seems to occur later in anterior sites than posterior sites. Correlational analysis showed a lower pattern of correlation between different frequencies in children than in young adults, suggesting a certain asynchrony in the maturation of different rhythms. The topographical analysis revealed similar topographies of the different rhythms in children and young adults. Principal Component Analysis (PCA) demonstrated the same internal structure for the Electroencephalogram of both age groups. Principal Component Analysis allowed to separate four subcomponents in the alpha range. All these subcomponents peaked at a lower frequency in children than in young adults. CONCLUSIONS: The present approaches complement and solve some of the incertitudes when the classical brain broad rhythm analysis is applied. Children have a higher absolute power than young adults for frequency ranges between 0-20 Hz, the correlation of Power Spectrum (PS) with age and the variance age comparison showed that there are six ranges of frequencies that can distinguish the level of EEG maturation in children and adults. The establishment of maturational order of different frequencies and its possible maturational interdependence would require a complete series including all the different ages. 相似文献
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Sovan Mitra Andreas Karathanasopoulos Georgios Sermpinis Christian Dunis John Hood 《European Journal of Operational Research》2015
The role of decision support systems in mitigating operational risks in firms is well established. However, there is a lack of investment in decision support systems in emerging markets, even though inadequate operational risk management is a key cause of discouraging external investment. This has also been exacerbated by insufficient understanding of operational risk in emerging markets, which can be attributed to past operational risk measurement techniques, limited studies on emerging markets and inadequate data. 相似文献