Site-specific detection of S-nitrosylated PKB alpha/Akt1 from rat soleus muscle using CapLC-Q-TOF(micro) mass spectrometry

Protein Kinase Balpha(PKBalpha, or Akt1) is believed to play a crucial role in programmed cell death, cancer progression and the insulin-signaling cascade. The protein is activated by phosphorylation at multiple sites and subsequently phosphorylates and activates eNOS. Free cysteine residues of the protein may capture reactive, endogenously produced nitric oxide (NO) as S-nitrosothiols. Site-specific detection of S-nitrosylated cysteine residues, usually at low stoichiometry, has been a major challenge in proteomic research largely due to the lack of mass marker for S-nitrosothiols that are very labile under physiologic conditions. In this report we describe a sensitive and specific MS method for detection of S-nitrosothiols in PKB alpha/Akt1 in rat soleus muscle. PKB alpha/Akt1 was isolated by immunoprecipitation and 2D-gel electrophoresis, subjected to in-gel tryptic digestion, and cysteinyl nitrosothiols were reacted with iodoacetic acids [2-C(12)/C(13) = 50/50] under ascorbate reduction conditions. This resulted in the production of relatively stable carboxymethylcysteine (CMC) immonium ions (m/z 134.019 and m/z 135.019) within a narrow argon collision energy (CE = 30 +/- 5 V) in the high MS noise region. In addition, free and disulfide-linked cysteine residues were converted to carboxyamidomethylcysteines (CAM). Tryptic S-nitrosylated parent ion was detected with a mass accuracy of 50 mDa for the two CMC immonium ions at the triggered elution time during capillary liquid chromatography (LC) separation. A peptide containing Cys(296) was discriminated from four co-eluting tryptic peptides under lock mass conditions (m/z 785.8426). S-nitrosothiol in the tryptic peptide, ITDFGLBKEGIK (B: CAM, [M + 2H](2+) = 690.86, Found: 690.83), is believed to be present at a very low level, since the threshold for the CMC immonium trigger ions was set at 3 counts/s in the MS survey. The high levels of NO that are produced under stress conditions may result in increased S-nitrosylation of Cys(296) which blocks disulfide bond formation between Cys(296) and Cys(310) and suppresses the biological effects of PKB alpha/Akt1. With the procedures developed here, this process can be studied under physiological and pathological conditions.     

化合物库的组合技巧和组合合成

从化合物库的组合技巧和组合合成方法两个方面探讨了组合化学及其在药物开发中的应用。     

RNA Methylation m6A: A New Code and Drug Target?

The m⁶A‐RNA modification is a dynamic and reversible process, which has emerged as a new RNA code for the regulation of gene expression. The functional network of methyltransferases (writers), demethylases (erasers), and binding proteins (readers) modulate the level of m⁶A modification. Dysfunction of RNA methylation has been associated with various fundamental biological processes and human diseases. Herein, we briefly introduce an understanding‐enabled manipulation on m⁶A‐RNA modification with an emphasis on the use of small‐molecule intervention.      

Open-access liquid chromatography/mass spectrometry in a drug discovery environment

The use of open-access mass spectrometry to monitor synthetic chemistry reactions, and also the integrity and purity of new chemical entities, has been a part of the medicinal chemist's tool-box for more than 5 years. Originally in our group at Wyeth Research there were two open-access methods available to the chemists, flow injection analysis (FIA) and liquid chromatography/mass spectrometry (LC/MS). The FIA method was approximately 3 min long, while the LC/MS method was approximately 20 min long (including an 8 min gradient). Within the first 2 years, the total number of open-access analyses increased by approximately 125%. It is interesting, however, that the number of LC/MS analyses increased by more than 285%. This is attributed to the fact that the chemists began using the LC/MS data to monitor reactions and also to check final product integrity and purity. In addition, the number of chemists performing parallel synthesis reactions has increased; thus, individual chemists can produce sample sets of up to 100 vials. This paper describes the implementation of new methodology, which accommodates the need for much faster run times and also the ability to acquire alternating positive and negative ion spectra within the same run. In addition, the instrument has been configured to e-mail the resulting processed data report to the submitting chemist. Several methods have been developed, including structure elucidation using in-source collision-induced dissociation (CID) and night-time analysis. The LC/MS methods for this system are described herein and are applicable to both industrial and academic synthetic chemistry optimization efforts.     

高校图书馆数字化建设中的特色服务

数字化时代，高校图书馆要利用自身优势，在数字化建设中做出自己的特色，即服务内容上突出学术性，大力发展多媒体型信息；服务方式上依托校园网，拓展数字化信息服务；服务形象上体现高素质，做好信息导航服务。     

试论现代图书馆的人文精神

通过对图书馆人文精神内涵及发展的阐述，指出了网络环境下图书馆的人文精神的严重缺失问题，从而提出了现阶段图书馆倡导人文精神的必要性及其具体实践途径。     

公共图书馆如何做好老年读者服务工作

老年读者服务工作是构建和谐社会的重要组成部分,为此,研究和分析了老年读者的心理特点和阅读特点,并提出图书馆做好老年读者服务工作的要点.     

浅谈开放教育与图书馆服务

广播电视大学图书馆是电大的文献信息中心,随着开放教育这一新的教育模式的出现,图书馆也应在整体上做出相应的改革,树立“以人为主体”的服务理念。未来图书馆事业发展的核心是信息增值服务和人本服务。     

北京-郑州运输通道内旅客出行的特征

为研究运输通道内客流变化规律,结合北京郑州铁路客运专线建设,运用简单抽样方法确定样本抽样率和样本容量,对通道内不同运输方式旅客出行调查数据进行统计分析。结果表明:出行旅客中企管人员最多;月收入小于1 000元的旅客主要选择公路出行,高于2 000元的旅客主要选择民航出行,介于二者之间的旅客多选择铁路出行;旅客多以出差、经商和探亲为出行目的;在小于300 km的出行距离内,公路占绝对优势;在300~1 500 km内,铁路优势明显;服务属性中,旅客最为关心的是安全,最不满意的是运行速度。     

基于人工神经网络的沥青路面剩余寿命分析

路面整体结构性能评估是公路养护或重建决策优化的一项重要参考依据，一般情况下，如果路面的服务能力指数降到可接受水平以下，就有必要对当前整体性能(剩余寿命)进行评价，决定是否需要延长寿命或者进行重建设计，因此，利用人工神经网络(ANN)方法建立了沥青路面剩余寿命预测模型，由此程序可以由落锤弯沉仪(FWD)弯沉数据快速预估路面的剩余寿命，这种方法直观、准确，并且不需要反算模量，对于道路工作者的养护和补强罩面工作具有参考价值。