基于生物质谱的蛋白质C末端研究进展

蛋白质的C末端在蛋白质进行各项生命活动过程中都起着极其重要的作用。它不仅标志着DNA转录翻译成蛋白质过程的初步完成,更是参与和调控了蛋白质的各种生理功能。研究蛋白质的C末端不仅有利于完整蛋白质的鉴定,对于在分子水平理解蛋白质的信号传导和生化功能是十分必要的。文中结合我们的研究工作,综述了近年来基于生物质谱的蛋白质C末端研究的相关进展,包括了C末端的识别、鉴定以及蛋白质C末端肽段富集的新方法和新技术。     

基于质谱的蛋白质N末端乙酰化程度定量方法的研究

随着质谱技术及各种定量方法的不断完善和发展,定量蛋白质组学的方法不断地被应用到各类生物学研究中。蛋白质组学定性定量数据的处理主要通过一些多功能的商业化或者开源软件来进行,如常用的数据分析软件Proteome Discoverer和Maxquant。但是在通过化学标记对蛋白质N末端乙酰化程度进行定量这一方面,Proteome Discoverer和Maxquant在一定程度上存在准确性不高和完整度不够的问题。于是本研究针对自己的实验特点,通过Java算法编写了相应的定量程序Acequant来完成N末端乙酰化程度的相对定量。本研究将该程序在已有相关报道的He La cell上进行了验证,Acequant共定量到1 587个蛋白质N末端,而Proteome Discoverer和Maxquant分别只定量到42个和306个N末端。同时,手动验证原始图谱也证实了Acequant定量的准确性更好。于是,本研究将此方法进一步应用到秀丽隐杆线虫N末端乙酰化的研究中,并初步发现了线虫整体的N末端乙酰化状态,为进一步的N末端研究提供了支持。     

微生物蛋白质组研究进展

本文概括性地叙述蛋白质组相关的基本概念,主要蛋白质组技术,微生物学蛋白质、比较蛋白质组、功能蛋白质组研究进展和蛋白质组技术在细胞微生物学研究中的应用。     

蛋白质基因组学：运用蛋白质组技术注释基因组

随着高通量DNA测序技术的飞速发展,越来越多的物种完成了基因组测序．定位编码基因、确定编码基因结构是基因组注释的基本任务,然而以往的基因组注释方法主要依赖于DNA及RNA序列信息．为了更加精确地解读完成测序的基因组,我们需要整合多种类型的组学数据进行基因组注释．近年来,基于串联质谱技术的蛋白质组学已经发展成熟,实现了对蛋白质组的高覆盖,使得利用串联质谱数据进行基因组注释成为可能．串联质谱数据一方面可以对已注释的基因进行表达验证,另一方面还可以校正原注释基因,进而发现新基因,实现对基因组序列的重新注释．这正是当前进展较快的蛋白质基因组学的研究内容．利用该方法系统地注释已完成测序的基因组已成为解读基因组的一个重要补充．本文综述了蛋白质基因组学的主要研究内容和研究方法,并展望了该研究方向未来的发展．     

蛋白质组研究技术及其进展

蛋白质组学是在后基因时代出现的一个新的研究领域．它是对机体或组织或细胞的全部蛋白质的表达和功能模式进行研究。介绍并总结了蛋白质组研究的主要技术,包括双向凝胶电泳、质谱技术、蛋白质芯片和生物信息学等。     

蛋白质组中蛋白质磷酸化研究进展

随着后基因组时代的到来 ,对生命体器官、组织或细胞的全部蛋白质的表达、修饰及相互作用的研究已成为蛋白质组学的重要任务。蛋白质磷酸化是细胞内信号转导和酶调控最常见的机制之一 ,人类基因组约 2 %的基因编码 5 0 0种激酶和 10 0种磷酸酶。蛋白质磷酸化和去磷酸化作为原核和真核细胞表达调控的关键环节 ,了解其对功能的影响可以深入理解生命系统在分子水平的调控状况。目前蛋白质组磷酸化研究仍是功能基因组面临的重大课题 ,本文对此作一综述     

蛋白质组与蛋白质组学

１９９４年澳大利有针对后基因组时代研究趋势,提出了蛋白质组和蛋白质组学的新概念。即把基因组所编码的所有蛋白为研究对象,直接探讨基因、蛋白的功能。其核心技术包括双向电泳、质谱分析、生物信息学等。该技术在探索疾病发生,寻找新药等方面取得越来越广泛的应用。     

叶绿体蛋白质组研究进展

亚细胞蛋白质组学是近年来蛋白组学研究中的一个热点。通过细胞器的纯化和亚细胞组分的分离,降低了样品的复杂性,增大了相应蛋白质组分的富集,有利于由此分离获得的蛋白质的序列分析及功能鉴定。叶绿体蛋白质组为植物亚细胞蛋白质组学研究中相对全面的一部分,利用亚细胞分离结合双向电泳技术系统地鉴定叶绿体中蛋白质组分是获取叶绿体蛋白质信息、确定其功能的重要技术手段。本文就近年来植物叶绿体蛋白质组涵盖的叶绿体内、外被膜、叶绿体基质、类囊体膜和类囊体腔蛋白的研究进行综述,以全面认识叶绿体蛋白的组成、特点及其在叶绿体生理生化代谢网络中的作用。     

定量蛋白质组同位素标记技术及应用

定量蛋白质组学是对蛋白质组进行精确的定量和鉴定的学科,突破了传统蛋白质组研究集中于对蛋白质的分离和鉴定,着重于定性定量解析细胞蛋白质的动态变化信息,更真实地反映了细胞功能、过程机制等综合信息。以同位素为内标的质谱分析新技术的提出,显示出可同时自动鉴定和精确定量的能力,代表了目前定量蛋白质组研究的主要发展方向。对近年来定量蛋白质组学同位素标记技术和应用研究所取得的重要进展以及最新的发展动态进行了综述。     

乳腺癌蛋白质组研究进展

乳腺癌是发生在乳房腺上皮组织的恶性肿瘤,其发病率占女性恶性肿瘤首位。近年来,蛋白质组研究广泛深入乳腺癌发病机理、临床早期诊断和治疗等各个方面。本文比较系统地综述了乳腺蛋白质组研究的样品处理、研究技术和研究成果。     

Evaluation of four phosphopeptide enrichment strategies for mass spectrometry-based proteomic analysis

Information about phosphorylation status can be used to prioritize and characterize biological processes in the cell. Various analytical strategies have been proposed to address the complexity of phosphorylation status and comprehensively identify phosphopeptides. In this study, we evaluated four strategies for phosphopeptide enrichment, using titanium dioxide (TiO₂) and Phos-tag ligand particles from in-gel or in-solution digests prior to mass spectrometry-based analysis. Using TiO₂ and Phos-tag magnetic beads, it was possible to enrich phosphopeptides from in-gel digests of phosphorylated ovalbumin separated by Phos-tag SDS-PAGE or in-solution serum digests, while minimizing non-specific adsorption. The tip-column strategy with TiO₂ particles enabled enrichment of phosphopeptides from in-solution digests of whole-cell lysates with high efficiency and selectivity. However, the tip-column strategy with Phos-tag agarose beads yielded the greatest number of identified phosphopeptides. The strategies using both types of tip columns had a high degree of overlap, although there were differences in selectivity between the identified phosphopeptides. Together, our results indicate that multi-enrichment strategies using TiO₂ particles and Phos-tag agarose beads are useful for comprehensive phosphoproteomic analysis.     

Mass spectrometry-based quantitative proteomic profiling.

Quantitative proteomics involves the identification and quantitation of protein components in various biological systems. Stable isotope labelling technology, by both metabolic and chemical methods, has been the most commonly used approach for global proteome-wide profiling. Recently, its capability has been extended from labelled pairs to multiple labels, allowing for the simultaneous quantification of multiplex samples. The ion intensity-based quantitative approach has progressively gained more popularity as mass spectrometry performance has improved significantly. Although some success has been reported, it remains difficult comprehensively to characterise the global proteome, due to its enormous complexity and dynamic range. The use of sub-proteome fractionation techniques permits a simplification of the proteome and provides a practical step towards the ultimate dissection of the entire proteome. Further development of the technology for targeting sub-proteomes on a functional basis - such as selecting proteins with differential expression profiles from mass spectrometric analyses, for further mass spectrometric sequencing in an intelligent manner--is expected in the near future.     

Current status of proteomic standards development

The generation of proteomic data is becoming ever more high throughput. Both the technologies and experimental designs used to generate and analyze data are becoming increasingly complex. The need for methods by which such data can be accurately described, stored and exchanged between experimenters and data repositories has been recognized. Work by the Proteome Standards Initiative of the Human Proteome Organization has laid the foundation for the development of standards by which experimental design can be described and data exchange facilitated. The Minimum Information About a Proteomic Experiment data model describes both the scope and purpose of a proteomics experiment and encompasses the development of more specific interchange formats such as the mzData model of mass spectrometry. The eXtensible Mark-up Language-MI data interchange format, which allows exchange of molecular interaction data, has already been published and major databases within this field are supplying data downloads in this format.     

Gold nanoparticles and bioconjugation: a pathway for proteomic applications

In the last few years gold nanoparticles (AuNPs) became extremely interesting materials due to their enhanced optical, chemical and electrical properties. With the intention of taking advantage of those properties, the use of AuNPs has spread into a wide variety of areas such as physics, chemistry, biology, industry and medicine. More interestingly, their ability to form robust conjugates with biomolecules has given proteomics a new tool to improve aspects where the current methods to study proteins and their interactions in living cells cannot achieve the success required. In this review we present some of the current methods for AuNPs synthesis, the tailoring of their surface with ligands to improve stability and strategies to conjugate with biomolecules. Lastly, we also discuss their application in proteomic methods and recent developments in clinical diagnosis.     

Separation methods for system-wide profiling of protein terminome

Protein N- and C-termini have specific biochemical properties and functions. They play vital roles in various biological processes, such as protein stability and localization. In addition, post-translational modifications and proteolytic processing generate different proteoforms at protein termini. In recent years, terminomics has attracted significant attention, and numerous strategies have been developed to achieve high-throughput and global terminomics analysis. This review summarizes the recent protein N-termini and C-termini enrichment methods and their application in different samples. We also look ahead further application of terminomics in profiling protease substrates and discovery of disease biomarkers and therapeutic targets.     

Differentially isotope-coded N-terminal protein sulphonation: combining protein identification and quantification

Most proteomic labelling technologies intend to improve protein quantification and/or facilitate (de novo) peptide sequencing. We present here a novel stable-isotope labelling method to simultaneously identify and quantify protein components in complex mixtures by specifically derivatizing the N-terminus of proteins with 4-sulphophenyl isothiocyanate (SPITC). Our approach combines protein identification with quantification through differential isotope-coded labelling at the protein N-terminus prior to digestion. The isotope spacing of 6 Da (unlabelled vs. six-fold 13C-labelled tag) between derivatized peptide pairs enables the detection on different MS platforms (MALDI and ESI). Optimisation of the reaction conditions using SPITC was performed on three model proteins. Improved detection of the N-terminally derivatized peptide compared to the native analogue was observed in negative-ion MALDI-MS. Simpler fragmentation patterns compared to native peptides facilitated protein identification. The 13C-labelled SPITC resulted in convenient peptide pair spacing without isotopic overlap and hence facilitated relative quantification by MALDI-TOF/TOF and LC-ESI-MS/MS. The combination of facilitated identification and quantification achieved by differentially isotope-coded N-terminal protein tagging with light/heavy SPITC represents, to our knowledge, a new approach to quantitative proteomics.     

ABRF-PRG04: differentiation of protein isoforms.

Accurate protein identification sometimes requires careful discrimination between closely related protein isoforms that may differ by as little as a single amino acid substitution or post-translational modification. The ABRF Proteomics Research Group sent a mixture of three picomoles each of three closely related proteins to laboratories who requested it in the form of intact proteins, and participating laboratories were asked to identify the proteins and report their results. The primary goal of the ABRF-PRG04 Study was to give participating laboratories a chance to evaluate their capabilities and practices with regards to sample fractionation (1D- or 2D-PAGE, HPLC, or none), protein digestion methods (in-solution, in-gel, enzyme choice), and approaches to protein identification (instrumentation, use of software, and/or manual techniques to facilitate interpretation), as well as determination of amino acid or post-translational modifications. Of the 42 laboratories that responded, 8 (19%) correctly identified all three isoforms and N-terminal acetylation of each, 16 (38%) labs correctly identified two isoforms, 9 (21%) correctly identified two isoforms but also made at least one incorrect identification, and 9 (21%) made no correct protein identifications. All but one lab used mass spectrometry, and data submitted enabled a comparison of strategies and methods used.     

Oxidative stress response: a proteomic view

The oxidative stress response is characterized by various effects on a range of biologic molecules. When examined at the protein level, both expression levels and protein modifications are altered by oxidative stress. While these effects have been studied in the past by classic biochemical methods, the recent onset of proteomics methods has allowed the oxidative stress response to be studied on a much wider scale. The input of proteomics in the study of oxidative stress response and in the evidence of an oxidative stress component in biologic phenomena is reviewed in this paper.