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61.
Sin-Hong Chen Ya-Chi Lin Ming-Kuei Shih Li-Fei Wang Shyh-Shyan Liu Jue-Liang Hsu 《Molecules (Basel, Switzerland)》2020,25(22)
Protein phosphorylation is a crucial post-translational modification that plays an important role in the regulation of cellular signaling processes. Site-specific quantitation of phosphorylation levels can help decipher the physiological functions of phosphorylation modifications under diverse physiological statuses. However, quantitative analysis of protein phosphorylation degrees is still a challenging task due to its dynamic nature and the lack of an internal standard simultaneously available for the samples differently prepared for various phosphorylation extents. In this study, stable-isotope dimethyl labeling coupled with phosphatase dephosphorylation (DM + deP) was tried to determine the site-specific degrees of phosphorylation in proteins. Firstly, quantitation accuracy of the (DM + deP) approach was confirmed using synthetic peptides of various simulated phosphorylation degrees. Afterwards, it was applied to evaluate the phosphorylation stoichiometry of milk caseins. The phosphorylation degree of Ser130 on α-S1-casein was also validated by absolute quantification with the corresponding synthetic phosphorylated and nonphosphorylated peptides under a selected reaction monitoring (SRM) mode. Moreover, this (DM + deP) method was used to detect the phosphorylation degree change of Ser82 on the Hsp27 protein of HepG2 cells caused by tert-butyl hydroperoxide (t-BHP) treatment. The results showed that the absolute phosphorylation degree obtained from the (DM + deP) approach was comparable with the relative quantitation resulting from stable-isotope dimethyl labeling coupled with TiO2 enrichment. This study suggested that the (DM + deP) approach is promising for absolute quantification of site-specific degrees of phosphorylation in proteins, and it may provide more convincing information than the relative quantification method. 相似文献
62.
Fang Hu Guobin Qi Kenry Duo Mao Shiwei Zhou Min Wu Wenbo Wu Bin Liu 《Angewandte Chemie (International ed. in English)》2020,59(24):9288-9292
Protected by the host cells, the hidden intracellular bacteria are typically difficult to kill by common antibiotics and cannot be visualized without complex cellular pretreatments. Herein, we successfully developed a bacteria‐metabolizable dual‐functional probe TPEPy‐d ‐Ala, which is based on d ‐alanine and a photosensitizer with aggregation‐induced emission for fluorescence turn‐on imaging of intracellular bacteria in living host cells and photodynamic ablation in situ. Once metabolically incorporated into bacterial peptidoglycan, the intramolecular motions of TPEPy‐d ‐Ala are inhibited, leading to an enhanced fluorescent signal, which allows the clear visualization of the intracellular bacteria. Moreover, TPEPy‐d ‐Ala can effectively ablate the labeled intracellular bacteria in situ owing to covalent ligation to peptidoglycan, yielding a low intracellular minimum inhibitory concentration (MIC) of 20±0.5 μg mL?1, much more efficient than that of a commonly used antibiotic, vancomycin. 相似文献
63.
Igor A. Kaltashov Anton El Khoury Chengfeng Ren Sergey N. Savinov 《Journal of mass spectrometry : JMS》2020,55(2)
Ruthenium is a platinoid that exhibits a range of unique chemical properties in solution, which are exploited in a variety of applications, including luminescent probes, anticancer therapies, and artificial photosynthesis. This paper focuses on a recently demonstrated ability of this metal in its +3 oxidation state to form highly stable complexes with tris (hydroxymethyl)aminomethane (H2NC(CH2OH)3, Tris‐base or T) and imidazole (Im) ligands, where a single RuIII cation is coordinated by two molecules of each T and Im. High‐resolution electrospray ionization mass spectrometry (ESI MS) is used to characterize RuIII complexes formed by placing a RuII complex [(NH3)5RuIICl]Cl in a Tris buffer under aerobic conditions. The most abundant ionic species in ESI MS represent mononuclear complexes containing an oxidized form of the metal, ie, [XnRuIIIT2 – 2H]+, where X could be an additional T (n = 1) or NH3 (n = 0‐2). Di‐ and tri‐metal complexes also give rise to a series of abundant ions, with the highest mass ion representing a metal complex with an empirical formula Ru3C24O21N6H66 (interpreted as cyclo(T2RuO)3, a cyclic oxo‐bridged structure, where the coordination sphere of each metal is completed by two T ligands). The empirical formulae of the binuclear species are consistent with the structures representing acyclic fragments of cyclo(T2RuO)3 with addition of various combinations of ammonia and dioxygen as ligands. Addition of histidine in large molar excess to this solution results in complete disassembly of poly‐nuclear complexes and gives rise to a variety of ionic species in the ESI mass spectrum with a general formula [RuIIIHiskTm (NH3)n ? 2H]+, where k = 0 to 2, m = 0 to 3, and n = 0 to 4. Ammonia adducts are present for all observed combinations of k and m, except k = m = 2, suggesting that [His2RuIIIT2 ? 2H]+ represents a complex with a fully completed coordination sphere. The observed cornucopia of RuIII complexes formed in the presence of histidine is in stark contrast to the previously reported selective reactivity of imidazole, which interacts with the metal by preserving the RuT2 core and giving rise to a single abundant ruthenium complex (represented by [Im2RuIIIT2 ? 2H]+ in ESI mass spectra). Surprisingly, the behavior of a hexa‐histidine peptide (HHHHHH) is similar to that of a single imidazole, rather than a single histidine amino acid: The RuT2 core is preserved, with the following ionic species observed in ESI mass spectra: [HHHHHH·(RuIIIT2)m ? (3m‐1)H]+ (m = 1‐3). The remarkable selectivity of the imidazole interaction with the RuIIIT2 core is rationalized using energetic considerations at the quantum mechanical level of theory. 相似文献
64.
65.
Nico Fleck Caspar A. Heubach Tobias Hett Florian R. Haege Pawel P. Bawol Helmut Baltruschat Olav Schiemann 《Angewandte Chemie (International ed. in English)》2020,59(24):9767-9772
The understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules, preferably acquired under native conditions. In this regard, pulsed dipolar EPR spectroscopy (PDS) in conjunction with site‐directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However, the currently available spin labels have diverse deficiencies for in‐cell applications, for example, low radical stability or long bioconjugation linkers. In this work, a synthesis strategy is introduced for the derivatization of trityl radicals with a maleimide‐functionalized methylene group. The resulting trityl spin label, called SLIM, yields narrow distance distributions, enables highly sensitive distance measurements down to concentrations of 90 nm , and shows high stability against reduction. Using this label, the guanine‐nucleotide dissociation inhibitor (GDI) domain of Yersinia outer protein O (YopO) is shown to change its conformation within eukaryotic cells. 相似文献
66.
Felix Nußbaumer Raphael Plangger Manuel Roeck Christoph Kreutz 《Angewandte Chemie (International ed. in English)》2020,59(39):17062-17069
We present the access to [5‐19F, 5‐13C]‐uridine and ‐cytidine phosphoramidites for the production of site‐specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5‐19F, 5‐13C]‐pyrimidine labels into five RNAs—the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ? (hHBV ?) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre‐microRNA (miRNA) 21 and the 59 nt full length pre‐miRNA 21. The main stimulus to introduce the aromatic 19F–13C‐spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole‐dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic 13C–19F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5‐19F, 5‐13C]/[5‐19F] pyrimidine labeling. For the 61 nt hHBV ? we find a beneficial 19F–13C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [19F, 13C]‐labeling of the SAM VI aptamer domain and the pre‐miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting. 相似文献
67.
以牛血清白蛋白和胰岛素为研究对象, 经1,5-I-AEDANS衍生后, 采用高效液相色谱(HPLC)进行分离分析, 发现两者的衍生存在明显的协同作用. 相似文献
68.
选取123例癫痫患者作为研究组,同期69例健康体检者作为对照组,均行三维动脉自旋标记技术(3D-ASL)灌注成像与静息态功能MRI(rs-FMRI)检查,研究二者在癫痫病诊断及鉴别中的价值.结果发现,研究组全面发作脑血流量(CBF)、低频振幅(ALFF)<部分发作和对照组,全面发作血清脑源性神经细胞营养因子(BDNF)... 相似文献
69.
量子点标记链霉亲和素及其生物活性检测 总被引:2,自引:0,他引:2
选用无机盐为前驱体,在水相中合成CdTe量子点,并用此量子点标记链霉亲和素,通过SephadexG-100层析分离纯化量子点标记的链霉亲和素,采用磁颗粒标记的链霉亲和素与量子点标记的链霉亲和素竞争结合辣根过氧化酶标记的生物素,即酶联免疫竞争抑制分析法检测链霉亲和素标记量子点后的生物活性,计算约70.3%的链霉亲和素标记到量子点上,且具有生物活性。每毫克量子点大约可偶联0.14 mg的链霉亲和素。采用荧光光谱研究量子点标记前后的荧光变化,标记后量子点的最大发射波长蓝移了8 nm,而发射光谱的半峰宽基本不变,说明量子点与链霉亲和素结合后粒子没有团聚,分散性好。 相似文献
70.
Alexandros P. SiskosAlison M. Hill 《Tetrahedron letters》2003,44(4):789-792
Labeled glycerol is a widely used biochemical probe to investigate biosynthetic pathways. A highly efficient synthesis of [1-13C, 18O]- and [1-13C, 2H2]-glycerol is described in which the 13C label is introduced using cyanide. The 18O label was introduced by a Pinner synthesis and reduction of the ester 5 allowed incorporation of the 2H labels. 相似文献