组蛋白H3赖氨酸27和36位甲基化修饰的鉴定

采用液相色谱-质谱联用技术结合生物信息学分析手段,研究Hela细胞组蛋白H3赖氨酸(Lysine (K))K27和K36位点带有甲基化和二甲基化修饰的多肽鉴定,通过二级质谱碎片解析和二级碎片丰度分析,对组蛋白H3赖氨酸K27和K36上甲基化和二甲基化修饰进行了鉴定和分析。     

蛋白质甲基化修饰的蛋白质组学分析方法新进展

蛋白质的甲基化修饰是一类重要的翻译后修饰。但与磷酸化、糖基化和泛素化等翻译后修饰相比,甲基化修饰的蛋白质组学分析方法开发还是一个较新的研究领域。近几年,由于甲基化修饰在表观遗传调控中的重要作用,这一修饰类型得到了越来越多的关注,相关的分析技术和分析方法也取得了较多进展。其中,基于质谱的蛋白质组学分析方法在甲基化修饰中发挥着关键的作用,实现了这一甲基化修饰的高通量分析。该综述将从甲基化修饰的分离富集、假阳性率控制以及定量蛋白质组学等方面对一些蛋白质甲基化修饰的分析技术和方法的最新进展进行介绍。     

DNA甲基化电化学分析

DNA甲基化是目前研究最多的DNA表观遗传修饰之一.大量研究表明,DNA甲基化会引起DNA结构、稳定性以及DNA与蛋白质相互作用方式的改变,从而影响基因表达,进而引起多种神经退行性疾病、免疫系统疾病甚至癌症.因此,发展简易、灵敏、准确、可靠的方法进行DNA甲基化的分析是至关重要的.本文简单介绍了DNA甲基化的分析方法,重点综述了DNA甲基化的电化学分析方法,并对DNA甲基化的研究前景进行了展望.     

羟乙基淀粉的羟乙基取代位置的研究

将羟乙基淀粉进行甲基化-水解-还原乙酰化反应，产生羟乙基葡萄糖的部分甲基化糖醇乙酸酯衍生物，应用气相色谱/化学电离质谱（GC/CIMS）和气相色谱/电子轰击质谱/质谱（GC/EIMS/MS）联用技术研究了羟乙基在淀粉糖环上的取代位置，发现G-2位取代的量是总取代量的82.0％。     

用于N-连接完整糖肽鉴定的糖基化蛋白质组学数据处理方法最新研究亮点北大核心CSCD

蛋白质糖基化与疾病的发生发展密切相关,临床上使用的大多数肿瘤标志物是糖基化蛋白质。在组学层次上进行位点特异性糖型的分析对发现新型疾病标志物、提高基于蛋白质糖基化的精准医学研究水平等具有重要作用。色谱-质谱联用技术在糖蛋白的分离分析研究中得到了广泛的应用。基于液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)的完整糖肽鉴定已成为研究蛋白质上位点特异性糖链修饰的主要手段,其主要优势在于分析过程中可以同时揭示蛋白位点与糖链修饰的信息,从而在组学层次实现规模化的蛋白糖基化分析。     

DNA甲基化修饰的定位分析方法研究进展

5?甲基胞嘧啶(5?Methylcytosine,5mC)作为DNA中研究最为广泛的表观遗传修饰,不改变基因的序列,但是可以调控基因表达,从而在生命体的生长、发育和疾病发生中发挥着重要作用.研究5mC的分布、变化及作用机制有助于加强对生命体活动本质的理解.阐明基因组DNA中5mC修饰的生物学功能主要依赖于精准破译其在基...     

《色谱原子光谱／质谱联用技术及形态分析》

元素形态分析是21世纪分析化学中发展最快的研究领域之一．而各种色谱（包括电泳）与原子光谱／质谱联用技术则是元素形态分析最有效的手段。本书详细介绍了自20世纪90年代以来联用技术及元素形态分析的研究成果及发展趋势。内容包括元素形态分析中的试样前处理技术、非色谱原子光谱／质谱联用技术、色谱（包括气相色谱、液相色谱、毛细管电泳）、原子光谱／质谱联用技术以及环境和生物体系中元素形态分析等。     

DNA加合物分析技术与方法

DNA加合物是由亲电性的小分子化合物与DNA链中的多种亲核性基团发生共价反应而形成的,可作为遗传毒性标志物.DNA加合物的生成是一些有毒化学品引起基因突变、发育畸形、肿瘤和癌症效应过程中的重要阶段,是医学、毒理学和环境健康研究的重要内容.发展快速、灵敏、准确的检测技术和方法是理解和研究DNA加合物生物学作用的关键.目前DNA加合物分析方法主要包括~(32)P后标记法、免疫分析法、免疫毛细管电泳-激光诱导荧光法、DNA测序以及色谱-质谱联用技术等.本文对这些技术和方法及其在DNA加合分析中的应用进行了综合评述,同时简要展望了DNA加合物分析技术和方法的发展.     

液相色谱-串联质谱法同时测定生物组织全基因组DNA甲基化和羟甲基化水平

测定全基因组DNA甲基化和羟甲基化水平对于研究环境污染物暴露的影响及致病机理具有重要的作用。本文建立了液相色谱-串联质谱（LC-MS/MS）同时测定动物组织中全基因组DNA甲基化和羟甲基化水平的方法。从动物组织样品中提取DNA,并将其酶解成单核苷,利用液相色谱-串联质谱法测定5-甲基胞嘧啶核苷、5-羟甲基胞嘧啶核苷和鸟嘌呤核苷的含量,计算全基因组DNA甲基化率和羟甲基化率。利用该方法研究了砷暴露对大鼠肝脏和小脑全基因组DNA甲基化和羟甲基化水平的影响,得到了砷影响DNA甲基化及羟甲基化的初步数据。该方法具有良好的重现性、灵敏度和稳定性,可以同时检测差异较大的DNA甲基化和羟甲基化水平。为同时研究DNA甲基化和羟甲基化水平提供了有力的支持。     

磷酸化蛋白质分析技术在蛋白质组研究中的应用

磷酸化修饰蛋白质的分析是蛋白质组研究中的重要内容。对于目前磷酸化蛋白质分析所涉及的各种方法,包括放射性同位素标记,抗体免疫印记等传统方法和以串联质谱各种扫描技术为基础,结合亲和提取、液相色谱-质谱联用等手段的新技术、新方法,以及这些技术在磷酸化蛋白质组学中的应用予以评述。     

液相色谱-串联质谱法测定生物样本全基因组DNA甲基化

建立了基于液相色谱-电喷雾串联质谱的分析方法,对生物样本中全基因组DNA甲基化水平进行定量测定.首先将DNA从生物样本中提取出来,将DNA片段酶解为单核苷,利用液相色谱-串联质谱测定胞嘧啶核苷和5-甲基胞嘧啶核苷的含量,从而计算出其全基因组DNA甲基化率.利用该法研究了暴露于全氟辛烷磺酸的L-02细胞、10例原发性肝癌病例血浆样本和10例对照血浆样本的全基因组DNA甲基化水平,得出了它们的总甲基化率变化的初步结果.本方法操作简单,具有很高的灵敏度和稳定性,为研究生物样本,尤其是临床上易得但DNA含量极低的血浆样本的总甲基化水平提供了思路.     

Determination of 5-hydroxymethyl-2′-deoxycytidine in Rice by High-performance Liquid Chromatography–Tandem Mass Spectrometry with Isotope Dilution

5-hydroxymethyl-2′-deoxycytidine (5-hmdC), which plays important roles in the epigenetic remodeling in mammals, has been recently determined in plant genomes at trace concentrations. In this study, a method for the sensitive, specific, and rapid assessment of 5-hmdC in rice is reported using high-performance liquid chromatography–tandem mass spectrometry (HPLC–tandem MS) with isotope dilution. The recovery of this method was from 75.6% to 81.3% for 5-hmdC and the intra- and inter-day precision values were within 5.81%. The quantification limit for 5-hmdC was 5.0?pM. Genomic DNA from rice was extracted and enzymatically digested for nucleotides for the HPLC–tandem MS assay. The 5-hmdC levels were 1.32?±?0.08, 1.64?±?0.12, and 1.98?±?0.07 modifications per million nucleosides in the Shengdao-18, Shengxiang-145, and Jindao-263 rice cultivars. The application of HPLC–tandem MS for 5-hmdC demonstrated its presence in the genomic DNA of rice and is useful in further studying the role of modified cytosine in the epigenetic regulation of rice.     

Epigenetics: an important challenge for ICP-MS in metallomics studies

Trace metal analysis has been long regarded as one of the principle tasks in areas of chemical analysis. At the early stage of instrumental development, total concentration was assessed in a variety of samples, yielding results, among others, for environmental, biological, and clinical samples. With the power of newer analytical techniques, such as inductively coupled plasma mass spectrometry (ICP-MS), accurate quantitative results can now be obtained at ultra-trace levels not only for metals, but also for metalloids and several non-metals. Even though the importance of trace elements in many biological processes is widely accepted, the elucidation of their biological pathways, understanding specific biological functions, or possible toxicological aspects is still a challenge and a driving force to further develop analytical methodology. Over the past decades, the scientific interest has moved from total element determination to include speciation analysis, which provides quantitative information of one or more individual element species in a sample. More recently, metallomics has been introduced as a more expanded concept, in which the global role of all metal/metalloids in a given system is considered. Owing to the multi-elemental focus of metallomics research, the use of ICP-MS becomes indispensable. Furthermore, considering the biological role of metals/metalloids and the use of elements as internal or external molecular tags, epigenetics should be considered as an important emerging application for metallomics studies and approaches. Among a variety of epigenetic factors, essential nutrients, but also environmental toxins, have been shown to affect DNA methylation, modification of histone proteins, and RNA interference, all of them being implicated in cancer, cardiovascular disease, and several inherited conditions. Recent studies suggest that epigenetics may be a critical pathway by which metals produce health effects. In this Trends article, the basic epigenetic concepts are introduced, followed by the early applications of ICP-MS classified as: (i) detection of ³¹P as a natural element tag for DNA, (ii) analysis of DNA adducts with metal-based drugs, (iii) element species as epigenetic factors.     

Dynamics and biological relevance of epigenetic N6-methyladenine DNA modification in eukaryotic cells

DNA methylation represents a major type of DNA modifications that play key roles in diverse biological processes. With the recent development of highly selective and sensitive bioanalytical techniques,N⁶-methyladenine（6mA） has been characterized as an important internal DNA modification dynamically occurring in multiple eukaryotes including humans. Increasing evidence has indicated that 6mA may act as a novel epigenetic modification involved in regulation of development, stress respon...     

Aspects of trapping efficiency and matrix effects in the development of a restricted‐access‐media‐based trap‐and‐elute liquid chromatography with mass spectrometry method

Online restricted access media with liquid chromatography and tandem mass spectrometry for the direct analysis of small molecules in biological fluids represents an interesting alternative to time‐demanding traditional sample preparation techniques. In this study, important considerations concerning the development of a restricted access media with liquid chromatography and tandem mass spectrometry method for the analysis of dansylated estrogens in biological matrix are presented. Parameters influencing peak tailing and trapping efficiency were evaluated. The key factors included the ion strength of the mobile phase, a loading flow rate of the sample onto the trap column, and selection of a proper stationary phase of the trap column for a given set of analytes. These parameters have proven to be essential for minimizing any unwanted chromatographic peak tailing. The bulk derivatization of the analytes in the biological fluids and its relationship to the observed matrix effects was evaluated as well.     

Toward a high-throughput approach to quantitative proteomic analysis: Expression-dependent protein identification by mass spectrometry

The isotope-coded affinity tag (ICAT) [1] technology enables the concurrent identification and comparative quantitative analysis of proteins present in biological samples such as cell and tissue extracts and biological fluids by mass spectrometry. The initial implementation of this technology was based on microcapillary chromatography coupled on-line with electrospray ionization tandem mass spectrometry. This implementation lacked the ability to select proteins for identification based on their relative abundance and therefore to focus on differentially expressed proteins. In order to improve the sample throughput of this technology, we have developed a two-step approach that is focused on those proteins for which the abundance changes between samples: First, a new software program for the automated quantification of ICAT reagent labeled peptides analyzed by microcapillary electrospray ionization time-of-flight mass spectrometry determines those peptides that differ in their abundance and second, these peptides are identified by tandem mass spectrometry using an electrospray quadrupole time-of flight mass spectrometer and sequence database searching. Results from the application of this approach to the analysis of differentially expressed proteins secreted from nontumorigenic human prostate epithelial cells and metastatic cancerous human prostate epithelial cells are shown.     

Determination of five nucleosides by LC‐MS/MS and the application of the method to quantify N6‐methyladenosine level in liver messenger ribonucleic acid of an acetaminophen‐induced hepatotoxicity mouse model

Ribonucleic acid N⁶‐methyladenosine methylation plays an important role in a variety of biological processes and diseases. Acetaminophen‐induced hepatotoxicity is one of the major challenges faced by clinicians. To date, the link between N⁶‐methyladenosine and acetaminophen‐induced hepatotoxicity has not been studied. In this study, a simple ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the simultaneous determination of five nucleosides (adenosine, uridine, cytidine, guanosine, and N⁶‐methyladenosine) in messenger ribonucleic acid. After enzymatic digestion of messenger ribonucleic acid, the nucleosides sample was separated on an Acquity UPLC column with gradient elution using methanol and 0.02% formic acid water, and detected by a Qtrap 4500 mass spectrometer with an electrospray ionization mode. The method was validated over the concentration ranges of 4–800 ng/mL for adenosine, uridine, cytidine, and guanosine and 0.1–20 ng/mL for N⁶‐methyladenosine. It was successfully applied to the determination of N⁶‐methyladenosine levels in liver messenger ribonucleic acid in an acetaminophen‐induced hepatotoxicity mouse model and a control group. This study offers a method for the determination of nucleoside contents in epigenetic studies and constitutes the first step toward the investigation of ribonucleic acid methylation in acetaminophen‐induced hepatotoxicity, which will facilitate the elucidation of its mechanism.     

Ion trap tandem mass spectrometric determination of F2-isoprostanes

A new tandem mass spectrometry (MS/MS) method based on the use of an ion trap mass spectrometer for the identification and quantitation of F(2)-isoprostanes has been developed. It consists of two solid-phase extractions and two derivation steps followed by gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS) analysis. This method is highly selective and sensitive and it has been successfully applied to biological samples.     

Mass spectrometry-based glycoprofiling of biopharmaceuticals by using an automated data processing tool: SimGlycan®

The therapeutic and immunological properties of biopharmaceuticals are governed by the glycoforms contained in them. Thus, bioinformatics tools capable of performing comprehensive characterization of glycans are significantly important to the biopharma industry. The primary structural elucidation of glycans using mass spectrometry is tricky and tedious in terms of spectral interpretation. In this study, the biosimilars of a therapeutic monoclonal antibody and an Fc-fusion protein with moderate and heavy glycosylation, respectively, were employed as representative biopharmaceuticals for released glycan analysis using liquid chromatography–tandem mass spectrometry instead of conventional mass spectrometry-based analysis. SimGlycan® is a software with proven ability to process tandem MS data for released glycans could identify eight additional glycoforms in Fc-fusion protein biosimilar, which were not detected during mass spectrometry analysis of released glycans or glyco-peptide mapping of the same molecule. Thus, liquid chromatography–tandem mass spectrometry analysis of released glycans not only complements conventional liquid chromatography–mass spectrometry-based glycan profiling but can also identify additional glycan structures that may otherwise be omitted during conventional liquid chromatography–tandem mass spectrometry based analysis of mAbs. The mass spectrometry data processing tools, such as PMI Byos™, SimGlycan^®, etc., can display pivotal analytical capabilities in automated liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry-based glycan analysis workflows, especially for high-throughput structural characterization of glycoforms in biopharmaceuticals.