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101.
Chemical probing represents a very versatile alternative for studying the structure and dynamics of substrates that are intractable by established high‐resolution techniques. The implementation of MS‐based strategies for the characterization of probing products has not only extended the range of applicability to virtually all types of biopolymers but has also paved the way for the introduction of new reagents that would not have been viable with traditional analytical platforms. As the availability of probing data is steadily increasing on the wings of the development of dedicated interpretation aids, powerful computational approaches have been explored to enable the effective utilization of such information to generate valid molecular models. This combination of factors has contributed to making the possibility of obtaining actual 3D structures by MS‐based technologies (MS3D) a reality. Although approaches for achieving structure determination of unknown targets or assessing the dynamics of known structures may share similar reagents and development trajectories, they clearly involve distinctive experimental strategies, analytical concerns and interpretation paradigms. This Perspective offers a commentary on methods aimed at obtaining distance constraints for the modeling of full‐fledged structures while highlighting common elements, salient distinctions and complementary capabilities exhibited by methods used in dynamics studies. We discuss critical factors to be addressed for completing effective structural determinations and expose possible pitfalls of chemical methods. We survey programs developed for facilitating the interpretation of experimental data and discuss possible computational strategies for translating sparse spatial constraints into all‐atom models. Examples are provided to illustrate how the concerted application of very diverse probing techniques can lead to the solution of actual biological systems. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
102.
Monika Hassel Michael G. Cornelius Jochen vom Brocke Heinz H. Schmeiser 《Electrophoresis》2010,31(2):299-302
The model organism Hydra has been used for molecular studies for more than 20 years, however, its DNA base composition has not been determined yet. We have analyzed DNA and total RNA of the freshwater polyp Hydra magnipapillata with two independent procedures of high accuracy and sensitivity – fluorescence labeling of nucleotides followed by CE‐LIF detection and 32P‐postlabeling. DNA of Hydra was digested either to deoxyribonucleoside‐5′‐monophosphates or deoxyribonucleoside‐3′‐monophosphates selectively conjugated with the fluorescent dye 4,4‐difluoro‐5,7‐dimethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene‐3‐propionyl ethylene diamine hydrochloride (BODIPY FL EDA) separated and detected using CE‐LIF. Both versions of the assay revealed a high A+T composition of 78 and 71%, whereas total DNA methylation (5‐methyldeoxycytidine) was 2.6 and 3.1%. Total Hydra RNA showed highest base levels for guanine (33%) and a level of 1.4% for pseudouracil. All values were in good agreement with those determined by the 32P‐postlabeling method. 相似文献
103.
Xu-Jie Xiong Hong WangWan-Bing Rao Xiao-Feng GuoHua-Shan Zhang 《Journal of chromatography. A》2010,1217(1):49-56
A BODIPY-based fluorescent derivatization reagent with a hydrazine moiety, 1,3,5,7-tetramethyl-8-aminozide-difluoroboradiaza-s-indacene (BODIPY-aminozide), has been designed for aldehyde labeling. An increased fluorescence quantum yield was observed from 0.38 to 0.94 in acetonitrile when it reacted with aldehydes. Twelve aliphatic aldehydes from formaldehyde to lauraldehyde were used to evaluate the analytical potential of this reagent by high performance liquid chromatography (HPLC) on C18 column with fluorescence detection. The derivatization reaction of BODIPY-aminozide with aldehydes proceeded at 60 °C for 30 min to form stable corresponding BODIPY hydrazone derivatives in the presence of phosphoric acid as a catalyst. The maximum excitation (495 nm) and emission (505 nm) wavelengths were almost the same for all the aldehyde derivatives. A baseline separation of all the 12 aliphatic aldehydes (except formaldehyde and acetaldehyde) is achieved in 20 min with acetonitrile–tetrahydrofuran (THF)–water as mobile phase. The detection limits were obtained in the range from 0.43 to 0.69 nM (signal-to-noise = 3), which are better than or comparable with those obtained by the existing methods based on aldehyde labeling. This reagent has been applied to the precolumn derivatization followed with HPLC determination of trace aliphatic aldehydes in human serum samples without complex pretreatment or enrichment method. 相似文献
104.
Sarkar R Concistrè M Johannessen OG Beckett P Denning M Carravetta M Al-Mosawi M Beduz C Yang Y Levitt MH 《Journal of magnetic resonance (San Diego, Calif. : 1997)》2011,212(2):460-463
The accurate temperature measurement of solid samples under magic-angle spinning (MAS) is difficult in the cryogenic regime. It has been demonstrated by Thurber et al. (J. Magn. Reson., 196 (2009) 84-87) [10] that the temperature dependent spin-lattice relaxation time constant of 79Br in KBr powder can be useful for measuring sample temperature under MAS over a wide temperature range (20–296 K). However the value of T1 exceeds 3 min at temperatures below 20 K, which is inconveniently long. In this communication, we show that the spin-lattice relaxation time constant of 127I in CsI powder can be used to accurately measure sample temperature under MAS within a reasonable experimental time down to 10 K. 相似文献
105.
106.
Ag-impurity effects on the first- and second-order quadrupole interaction (QI) at 23Na site in an isomorphic mixed system, Na1−xAgxNO2 (x=0, 0.0084, 0.026, 0.079, 0.094, 0.16), have been investigated by employing 23Na (I=3/2) magic angle spinning nuclear magnetic resonance (MAS NMR) technique. The central transition (CT) and satellite transition (ST) are simultaneously observed with this system. From the spectral analysis, the quadrupole parameter and its distribution width are obtained as a function of Ag concentration. From the intensity loss of CT MAS centerband and of the envelope function of ST MAS sidebands due to impurities, the range of their influence on the second- and first-order QI is estimated. The estimated ranges contain the second and first neighbouring Na sites from the resonating 23Na nucleus for the first- and second-order QI, respectively. 相似文献
107.
Tamás KálaiMark R. Fleissner József Jek?Wayne L. Hubbell Kálmán Hideg 《Tetrahedron letters》2011,52(21):2747-2749
New pyrroline nitroxides attached to a terminal acetylenic sulfone, a dibenzocyclooctyne or a cyclooctyne carboxylic acid were synthesized and tested in Cu-free click reactions to conjugate these new spin labels with 4-azido-TEMPO, azidophenylalanine and an azidophenylalanine-containing protein. 相似文献
108.
109.
110.
Alberto Paradela Miguel Marcilla Laura Ferreira Marisol Fernández Francisco García-del Portillo 《Talanta》2010,80(4):1496-3398
An evaluation of the ICPL (isotope-coded protein labeling) non-isobaric labeling technique was performed using two different biological models. Two samples containing phage T4 capsids were mixed in a 1:1 ratio after being labeled with the light or heavy versions of the ICPL reagent. The analysis of this proteome demonstrated the feasibility of this approach for differential quantitative proteomics and was employed to optimize the experimental parameters of the ICPL workflow. ICPL-mediated analysis of two more complex proteomes, those of a Salmonella enterica serovar Typhimurium virulent strain and an isogenic attenuated mutant, and its comparison with the results obtained in a 2D-PAGE “classical” approach confirmed that ICPL is a valuable alternative to other labeling techniques currently in use. In addition, our results suggest that labeling at the peptide level instead of following the standard ICPL workflow should increase both the number of proteins quantified and the reliability of the quantification. 相似文献