Label-free relative quantification of co-eluting isobaric phosphopeptides of insulin receptor substrate-1 by HPLC-ESI-MS/MS

Intracellular signal transduction is often regulated by transient protein phosphorylation in response to external stimuli. Insulin signaling is dependent on specific protein phosphorylation events, and analysis of insulin receptor substrate-1 (IRS-1) phosphorylation reveals a complex interplay between tyrosine, serine, and threonine phosphorylation. The phosphospecific antibody-based quantification approach for analyzing changes in site-specific phosphorylation of IRS-1 is difficult due to the dearth of phospho-antibodies compared with the large number of known IRS-1 phosphorylation sites. We previously published a method detailing a peak area-based mass spectrometry approach, using precursor ions for peptides, to quantify the relative abundance of site-specific phosphorylation in the absence or presence of insulin. We now present an improvement wherein site-specific phosphorylation is quantified by determining the peak area of fragment ions respective to the phospho-site of interest. This provides the advantage of being able to quantify co-eluting isobaric phosphopeptides (differentially phosphorylated versions of the same peptide), allowing for a more comprehensive analysis of protein phosphorylation. Quantifying human IRS-1 phosphorylation sites at Ser303, Ser323, Ser330, Ser348, Ser527, and Ser531 shows that this method is linear (n = 3; r² = 0.85 ± 0.05, 0.96 ± 0.01, 0.96 ± 0.02, 0.86 ± 0.07, 0.90 ± 0.03, 0.91 ± 0.04, respectively) over an approximate 10-fold range of concentrations and reproducible (n = 4; coefficient of variation = 0.12, 0.14, 0.29, 0.30, 0.12, 0.06, respectively). This application of label-free, fragment ion-based quantification to assess relative phosphorylation changes of specific proteins will prove useful for understanding how various cell stimuli regulate protein function by phosphorylation.     

197AU,57Fe and121Sb Mössbauer study of gold minerals and ores

A survey of the¹⁹⁷Au Mössbauer spectra of naturally occurring gold species is given. Gold minerals have been studied as natural specimens or as synthetic analogues. Gold impurities have been identified in pyrites and arsenopyrites. An example of the use of¹²¹Sb, and⁵⁷Fe Mössbauer spectroscopy in characterizing gold-bearing ore minerals is given.     

New99mTc-diiodine substituted IDA derivative (DIIODIDA) for hepatobiliary imaging

A new diiodine substituted IDA derivative, 2,4-diiodine-6-methyl IDA (DIIODIDA) was synthesized and labeled with^99mTc. It was established that^99mTc-DIIODIDA had high radiochemical purity. Biodistribution and influence of bilirubin on^99mTc-DIIODIDA biokinetics has been studied in rats and compared to the corresponding results for^99mTc-SOLCOIODIDA. Related to^99mTc-SOLCOIODIDA,^99mTc-DIIODIDA has much better biliary exretion (55.18 versus 43.63%). No change of^99mTc-DIIODIDA biokinetics, under influence of bilirubin was noticed. Biliary excretion of^99mTc-SOLCOIODIDA has been reduced for about 60%. The protein binding of^99mTc-DIIODIDA and^99mTc-SOLCOIODIDA were also determined, using in vitro method of precipitation. These results showed that^99mTc-DIIODIDA hepatobiliary imaging agent is superior to the presently used^99mTc-monoiodine IDA derivatives.     

一般n阶特征量泛函的Euler-Lagrange方程及与定量因果原理、相对性原理和广义牛顿三定律的统一

本文获得了有各种相互作用的一般n阶特征量泛函,其耦合系数反映了不同特征量泛函之间的耦合强度.依据定量因果原理,导出了一般n阶特征量泛函的变分原理,获得了一般n阶特征量泛函的Euler-Lagrange方程,它的不同系数可拟合不同的物理现实,如从线性到任意n阶非线性物理系统,使复杂难解的任意n阶非线性物理系统变得具体可解.并获得了该对称变换下不变的m个的守恒量,以及它们之间的关系和统一描述.依据定量因果原理导出了相对性原理,证明了绝对加速参考系、牵连参考系和相对参考系的力都有来自加速度和质量变化的贡献.利用定量因果原理自然导出了广义牛顿第一定律和广义牛顿第二定律,而且还导出了一个新定律,即广义牛顿第三定律,亦即平移不变性系统合力为零定理.进而将研究结论应用于对银河系的修正引力势、分子势、夸克禁闭势等,且其结果与物理实验一致.     

On relative stability of linear stationary feedback control systems with time delay

A method is presented whereby the absolute and relative stabilityof linear control systems containing transport lag can be determined.As a result feedback systems with variable time delay and loopgain, may be investigated in straightforward manner.     

Radiation-induced electron paramagnetic resonance signal and soybean isoflavones content

Electron Paramagnetic Resonance (EPR) is a well-known spectroscopic technique that detects paramagnetic centers and can detect free radicals with high sensitivity. In food, free radicals can be generated by several commonly used industrial processes, such as radiosterilization or heat treatment. EPR spectroscopy is used to detect radioinduced free radicals in food. In this work the relation between EPR signal induced by gamma irradiation treatment and soybean isoflavones content was investigated. Present results did not show correlation between total isoflavones content and the EPR signal. Nevertheless, some isoflavone contents had a negative correlation with the radiation-induced EPR signal.     

Quantification of phosphorylation of insulin receptor substrate-1 by HPLC-ESI-MS/MS

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) regulates the function and subsequent insulin signaling of this protein. Human IRS-1 has 1242 amino acid residues, including 182 serines and 60 threonines. The size, complexity, and relatively low abundance of this protein in biological samples make it difficult to map and quantify phosphorylation sites by conventional means. A mass spectrometry peak area based quantification approach has been developed and applied to assess the relative abundance of IRS-1 phosphorylation in the absence or presence of stimuli. In this method, the peak area for a phosphopeptide of interest is normalized against the average of peak areas for six selected representative IRS-1 peptides that serve as endogenous internal standards. Relative quantification of each phosphopeptide is then obtained by comparing the normalized peak area ratios for untreated and treated samples. Two non-IRS-1 peptides were added to each digest for use as HPLC retention time markers and additional standards as well as references to the relative quantity of IRS-1 in different samples. This approach does not require isotopic or chemical labeling and can be applied to various cell lines and tissues. Using this method, we assessed the relative changes in the quantities of two tryptic phosphopeptides isolated from human IRS-1 expressed in L6 cells incubated in the absence or presence of insulin or tumor necrosis factor-alpha. Substantial increases of phosphorylation were observed for Thr(446) upon stimulation. In contrast, no obvious change in the level of phosphorylation was observed for Ser(1078). This mass spectrometry based strategy provides a powerful means to quantify changes in the relative phosphorylation of peptides in response to various stimuli in a complex, low-abundance protein.     

Observation of quantum coherence in mesoscopic molecular magnets

By applying a transverse magnetic field B( perpendicular) of sufficient strength to the uniaxial molecular magnets Fe8 and Mn12, the tunneling splitting Delta(t) of their S = +/-10 magnetic ground states can be made large compared to perturbations such as hyperfine and dipolar interactions. We present evidence for such a Delta(t) from magnetic specific heat data below 1 K that is consistent with coherent quantum mechanical tunneling in a "mesoscopic" system under such conditions.