MEWS

针对蛋白质组学研究中^18O标记存在的一些问题，北京放射与辐射医学研究所、蛋白质组研究中心、蛋白质组学国家重点实验室钱小红研究小组创新性地提出了一种基于酸酐双功能试剂修饰辅助的稳定同位素^18O标记的蛋白质组双重定量新方法。     

定量蛋白质组学新技术新方法研究获新进展

9月3日,中科院大连化学物理研究所邹汉法、叶明亮研究员带领的团队,在定量蛋白质组学新技术新方法方面取得新进展,发展了一种基于胰蛋白酶催化的N端稳定同位素编码的氨基酸标记的相对定量蛋白质组学方法。相关研究成果发表在德国《应用化学》上。蛋白质组学研究的主要内容是蛋白质的表达水平,翻译后修饰,蛋白与蛋白相互作用等,最终的     

组蛋白翻译后修饰无标定量方法可靠性的比较

组蛋白翻译后修饰是一种表观遗传学修饰,参与调控细胞的新陈代谢等重要生理过程。蛋白质组学发展迅速,使监控组蛋白翻译后修饰的动态变化成为可能。目前主要有3种无标定量方法（谱图计数法、峰面积积分法和信号强度法）,但何种定量方法更可靠尚未见系统性的详细报道。在稳定同位素标记细胞培养技术（SILAC）基础上,对去乙酰化酶抑制剂（SAHA）调控细胞乙酰化修饰水平的定量数据进行对比,比较3种无标定量方法对组蛋白翻译后修饰进行的定量分析,利用定量结果的标准差（SD）评估定量的可靠性,最终发现基于峰面积积分法定量的结果可靠性最高。该研究对难以进行同位素标记实验的样本分析,尤其对临床样本、大样本的组蛋白修饰谱分析具有重要参考意义。     

基于稳定同位素标记与质谱分析的蛋白质定量算法研究进展

蛋白质定量研究已成为蛋白质组学的热点,它是疾病相关生物标志物发现的重要途径.基于稳定同位素标记的质谱分析技术是蛋白质定量最常用的方法之一.随着实验方法的发展和改进,定量数据处理方法也在不断更新与完善.一般来说,定量数据处理包括四步:搜库鉴定、图谱定量信息提取与计算、肽段丰度比计算和蛋白质丰度比计算及差异显著性分析,其中后三步是数据处理的核心.目前,后三步中每步都有多种可选算法,这些算法一般都是针对特定实验技术而提出的,缺乏深入的工作对它们进行系统比较和优化.为此,在总结目前主要实验技术方法的基础上,论述了定量算法的现状和存在问题,并针对一些问题提出了可行的解决办法.     

NEWS——蛋白质组18O双重定量新方法

针对蛋白质组学研究中18O标记存在的一些问题，北京放射与辐射医学研究所、蛋白质组研究中心、蛋白质组学国家重点实验室钱小红研究小组创新性地提出了一种基于酸酐双功能试剂修饰辅助的稳定同位素18O标记的蛋白质组双重定量新方法。该研究发表在Analytical Chemistry，ASAP Ar     

基于生物质谱的乙酸酐稳定同位素标记定量蛋白质组学方法的建立与优化

建立了一种基于生物质谱的乙酸酐稳定同位素标记,定量蛋白质组学研究方法,优化了影响标记效率的各种条件。在pH8.0的Na2B4O7/H3BO3缓冲体系中,当乙酸酐摩尔浓度25倍过量于肽段摩尔量,22℃反应30 min时,标记即可完全。对多对H6/D6-乙酸酐标记肽段在基质辅助激光解吸电离质谱中的动态范围及定量准确度进行了考察,并通过串联质谱分析确定了乙酰化位点。结果表明:在10倍和30倍动态范围内,线性关系良好(r=0.99,r=0.98),理论值和观测值的偏差分别为0.5%和20%。     

基于生物质谱的乙酸酐稳定同位素标记

建立了一种基于生物质谱的乙酸酐稳定同位素标记,定量蛋白质组学研究方法,优化了影响标记效率的各种条件.在pH 8.0的Na2B4O7/H3BO3 缓冲体系中,当乙酸酐摩尔浓度25倍过量于肽段摩尔量,22℃反应30 min时,标记即可完全.对多对H6/D6-乙酸酐标记肽段在基质辅助激光解吸电离质谱中的动态范围及定量准确度进行了考察,并通过串联质谱分析确定了乙酰化位点.结果表明: 在10倍和30倍动态范围内,线性关系良好(r=0.99, r=0.98),理论值和观测值的偏差分别为0.5%和20%.     

等电聚焦分离与18O稳定同位素标记联用的磷酸化蛋白质组学定量方法研究

磷酸化蛋白质组学定量分析,要对磷酸化修饰富集技术和定量技术进行研究。基于此,本研究采用18O稳定同位素标记技术对胰蛋白酶酶解肽段混合物进行标记,并对其标记时间和标记后胰蛋白酶的变性条件进行优化。结果表明:在pH=4～5的KH2PO4缓冲体系中,37℃,标记反应持续19～24h,除了C-端肽之外,几乎所有的肽段都可达到100%标记;采用TCEP可以有效地抑制16O-18O回标现象。建立了与18O标记技术兼容性良好的IPG-IEF技术对磷酸化肽段进行选择性富集,富集后共从HepG2细胞中鉴定到491个磷酸化位点、362个磷酸化肽段和356个磷酸化蛋白,表明IPG-IEF在大规模磷酸化肽段分离富集中是有效的;最后与高准确度高灵敏度高分辨率的LTQ-FTICR质谱仪联用,建立了基于18O-IPG-IEF-LTQ-FTICR的磷酸化蛋白质组定量技术。实验结果表明,该技术可以实现磷酸化肽段的有效定性和定量。本研究为磷酸化蛋白质组学定量研究提供了实用技术。     

基于多级质谱的蛋白质组定量新方法新技术进展

定量蛋白质组学已经成为蛋白质组学的一个重要分支,以生物质谱为核心的定量蛋白质组方法日益发展。按照定量所依据的质谱信号来源于一级质谱谱图还是多级质谱谱图可以将定量蛋白质组方法分为一级质谱定量和多级质谱定量。本文主要综述基于多级质谱的定量方法和技术进展,分析比较了这些方法的优缺点,并对基于多级质谱的定量方法发展进行了展望。     

用EM算法改进鸟枪法蛋白质鉴定中的无标记定量方法

蛋白质定量是探索疾病发生发展状况和寻找新药靶标的重要手段。在shotgun蛋白组学中,目前常用定量方法包括综合同位素标记后的质谱峰强度方法和无标记定量方法。根据数据类型无标记定量方法可以分为两类:基于鉴定蛋白的质谱数的方法和基于质谱峰强度的方法。本研究主要用EM算法改进基于鉴定蛋白质谱数的定量方法,并用免疫印迹实验获得的酵母全蛋白的丰度来验证EM算法改进后定量的有效性结果表明,改进后的质谱数和蛋白丰度的相关性比改进前有一定的提高。同时,利用这些数据对主要的几种基于鉴定蛋白的质谱数的模型进行了比较,发现PAI模型最好,SpS模型次之,emPAI模型最不适合于蛋白质定量。     

比较蛋白质组学研究中的稳定同位素标记技术

比较蛋白质组学是指在蛋白质组学水平上研究正常和病理情况下细胞或组织中蛋白质表达变化,以期发现具有重要功能的生物标识物,为疾病的早期诊断提供依据。近年来它正成为蛋白质组学研究的热点和发展趋势。比较蛋白质组学的研究方法和策略有多种,本文就最近几年来稳定同位素标记技术(体内代谢标记技术和体外化学标记技术)在比较蛋白质组学研究中的进展进行综述。     

Applications of stable isotope dimethyl labeling in quantitative proteomics

Mass spectrometry has proven to be an indispensable tool for protein identification, characterization, and quantification. Among the possible methods in quantitative proteomics, stable isotope labeling by using reductive dimethylation has emerged as a cost-effective, simple, but powerful method able to compete at any level with the present alternatives. In this review, we briefly introduce experimental and software methods for proteome analysis using dimethyl labeling and provide a comprehensive overview of reported applications in the analysis of (1) differential protein expression, (2) posttranslational modifications, and (3) protein interactions.     

Quantitative proteomics for cancer biomarker discovery

The mass spectrometry (MS)-based quantitative proteomics is powerful to discover disease biomarkers that can provide diagnostic, prognostic and therapeutic targets, and it also can address important problems in clinical and translational medical research. The current status of MS-based quantification strategy and technical advances of several main quantitative assays (two-dimensional (2-D) gel-based methods, stable isotope labeling with amino acids in cell culture (SILAC), isotope-coded affinity tag (ICAT), the isobaric tags for relative and absolute quantification (iTRAQ), 1?O labeling, absolute quantitation and label-free quantitation) have been summarized and reviewed. At present, except 2-D gel-based methods, several stable isotope labeling quantitative techniques, including SILAC, ICAT and iTRAQ, etc, have been widely applied in identification of differential expression of proteins, post-translational modifications and protein-protein interactions in order to look for novel candidate cancer biomarkers from different physiological states of cells, body fluids or tissue samples. Also, the advantages and challenges of different quantitative proteomic approaches are discussed in identification and validation of candidate targets.     

MeCAT—new iodoacetamide reagents for metal labeling of proteins and peptides

Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metal-coded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labeling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent.     

Quantitative top-down proteomics of SILAC labeled human embryonic stem cells

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells with relevance to treatment of numerous medical conditions. However, a global understanding of the role of the hESC proteome in maintaining pluripotency or triggering differentiation is still largely lacking. The emergence of top-down proteomics has facilitated the identification and characterization of intact protein forms that are not readily apparent in bottom-up studies. Combined with metabolic labeling techniques such as stable isotope labeling by amino acids in cell culture (SILAC), quantitative comparison of intact protein expression under differing experimental conditions is possible. Herein, quantitative top-down proteomics of hESCs is demonstrated using the SILAC method and nano-flow reverse phase chromatography directly coupled to a linear-ion-trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FT-ICR-MS). In this study, which to the best of our knowledge represents the first top-down analysis of hESCs, we have confidently identified 11 proteins by accurate intact mass, MS/MS, and amino acid counting facilitated by SILAC labeling. Although quantification is challenging due to the incorporation of multiple labeled amino acids (i.e., lysine and arginine) and arginine to proline conversion, we are able to quantitatively account for these phenomena using a mathematical model.     

Quantitative mass spectrometry in proteomics: a critical review

The quantification of differences between two or more physiological states of a biological system is among the most important but also most challenging technical tasks in proteomics. In addition to the classical methods of differential protein gel or blot staining by dyes and fluorophores, mass-spectrometry-based quantification methods have gained increasing popularity over the past five years. Most of these methods employ differential stable isotope labeling to create a specific mass tag that can be recognized by a mass spectrometer and at the same time provide the basis for quantification. These mass tags can be introduced into proteins or peptides (i) metabolically, (ii) by chemical means, (iii) enzymatically, or (iv) provided by spiked synthetic peptide standards. In contrast, label-free quantification approaches aim to correlate the mass spectrometric signal of intact proteolytic peptides or the number of peptide sequencing events with the relative or absolute protein quantity directly. In this review, we critically examine the more commonly used quantitative mass spectrometry methods for their individual merits and discuss challenges in arriving at meaningful interpretations of quantitative proteomic data.     

蛋白质组学定量的技术与方法研究进展

蛋白质组学是一门在整体水平上研究细胞内蛋白质组成及其活动规律的新兴学科。定量蛋白质组学是指通过某种方法或技术，对生物样品(细胞、组织或体液等)在某些过程中蛋白质的含量进行比较分析。近几年来，定量蛋白质组的技术发展很快，稳定同位素标记技术的提出，为准确定量在细胞或组织体系中发挥重要功能的低丰度蛋白质提供了一个理想的方法。本文综述了蛋白质组定量分析技术及其最新的研究进展。