人乳中蛋白质的组成分析

在对人乳样品分离与纯化的基础上,采用“Bottom Up”并结合高效液相色谱（HPLC）和配有纳米喷雾（Nano-spray）技术的高分辨傅里叶变换离子回旋共振质谱（FT-ICR-MS）,将人乳各部分蛋白质样品酶解成多肽片段。 利用碰撞活化解离（CAD）和电子捕获解离（ECD）2种解离方式断裂机理的互补性规律,借助Mascot软件数据库,快速分析了人乳样品中乳脂部分、乳清部分和乳粒部分所含主要蛋白质的组成。人乳样品各部分的共同物性是均含有多种角蛋白、乳白蛋白、乳铁蛋白,但含量和种类各有不同,这既表明了人乳特殊的营养成分,又从另一角度显示出人乳各个部分营养价值的差异。     

金属氧化物在蛋白质组学研究中的应用

本文对近年来金属氧化物在蛋白质组学研究中发挥的作用进行了综述。重点介绍了该类化合物在蛋白质样品富集、固定化酶反应器、生物传感器和生物移植材料等方面的应用,并展望了其在蛋白质组学研究中的发展前景。     

亲和层析在蛋白质研究中的应用进展

文中介绍了生物、免疫、固定化金属离子拟生物几类常规亲和层析的原理及其在蛋白质分离纯化以及分析鉴定方面的应用（引用文献25篇）。     

结构蛋白质组学研究进展

蛋白质结构与其生物学功能直接相关，蛋白质功能的调控也主要依赖于其构象和相互作用的动态调节。对蛋白质结构和功能的研究一直是生命科学领域的研究热点，也是当前蛋白质组学研究的重要发展方向。该综述重点讨论了近年来基于质谱的结构蛋白质组学主要分析方法的原理、进展和应用，主要包括非变性质谱法、限制性蛋白质酶切法、化学交联法、氢氘交换法、共价化学标记法、热稳定性分析法等；最后对结构蛋白质组学的发展进行了总结与展望。     

色谱技术在蛋白质组学研究中的应用引言

蛋白质组是指一个有机体的基因组所表达的全部蛋白质。蛋白质组学是研究有机体蛋白质的组成及其变化规律的科学。蛋白质组成的高度复杂性和随时间、空间变化的特点,对蛋白质组的研究技术和方法提出了巨大挑战。色谱作为现代分离科学的核心技术之一,在蛋白质组研究中发挥了重要作用。我们可以通过对组织、细胞或体液中成千上万种蛋白质/多肽的色谱预分离,降低样本的复杂程度,提高蛋白质的鉴定率;我们可以通过亲和色谱对翻译后修饰的蛋白质/多肽进行特异性富集分离,去除非修饰的蛋白质/多肽,实现修饰蛋白的成功鉴定;我们还可以通过色谱 质谱联用技术,获得蛋白质/多肽在色谱分离中的保留时间或峰面积,实现蛋白质的规模化定量与鉴定等。 为了集中展示我国科学家在色谱技术及其在蛋白质组学研究中的应用方面所取得的成果,《色谱》杂志特此在2010年第2期编辑出版了“色谱技术在蛋白质组学研究中的应用”专栏。我们邀请了在该领域具有突出成绩或学术造诣的部分专家、学者撰写了相关的学术论文和综述。希望通过这些文章所介绍的工作,为进一步提高色谱技术在蛋白质组学研究中的应用水平,推动我国蛋白质组学的发展和取得创新性的研究成果作出贡献。     

表面等离子体共振技术在蛋白质-蛋白质相互作用研究中的应用

表面等离子共振(SPR)近年来迅速发展为用于分析生物分子相互作用的一项技术.该技术无需标记、特异性强、灵敏度高、样品用量小,可实现在线连续实时检测.目前SPR已被广泛应用于免疫学、蛋白质组学、药物筛选、细胞信号转导、受体/配体垂钓等领域.该文阐述了基于表面等离子体共振技术生物传感器的基本原理和技术流程,综述了SPR在蛋白质-蛋白质相互作用动力学研究、蛋白质结构及功能研究、蛋白质突变和碎片分析、信号转导中的应用以及SPR在蛋白质-蛋白质相互作用研究中的多项关键技术.指出SPR通过与光谱、电化学等多技术联用后,可以获得更加详实的信息.     

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。     

功能材料在蛋白质组研究中的应用进展

随着对蛋白质组鉴定深度、定量准确性及分析速度越来越高的要求,对蛋白质组学方法的研发提出了新的挑战。为了应对这些挑战,传统的蛋白质组学方法因其灵敏度低、准确性差以及耗时长等不足已经难以满足蛋白质组学研究领域不断提出的新需求。而将通过光、电、磁、热、化学、生化等作用合成具有特定功能的材料用于蛋白质组的研究,可以克服传统蛋白质组学分析技术的局限性,为蛋白质组学研究起到促进作用。该文对功能材料在蛋白质组研究中应用的新进展进行综述。     

蛋白质组学研究中的色谱分离技术

多维色谱-质谱技术正逐渐成为蛋白质组学研究的重要技术平台。对该技术及其应用的最新发展动态进行了综述,共引用文献59篇。     

蛋白质组学分析中蛋白质快速酶解方法的研究进展

蛋白质组学技术通过分析生物体内实现功能活动的蛋白质,帮助人们深入理解生物过程中的分子机制。基于生物质谱的自下而上鸟枪法蛋白质组学技术在鉴定深度和广度方面均显示出独特的优势,但其样品前处理流程复杂,过夜的蛋白质酶解反应步骤制约了样品前处理的效率。探索能够显著加快酶解反应速度的方法能够提高蛋白质组学分析的效率,以应对大规模、高通量的分析需求。文章综述了蛋白质快速酶解方法的研究进展,按照加速反应策略的不同,对引入额外能量、缩小反应体系体积和改变酶试剂形态三类工作分别展开介绍并总结其优缺点,展望了蛋白质组学快速样品前处理的发展前景。     

Secretory immunoglobulin a from human milk catalyzes milk protein phosphorylation

This article presents evidence that protein kinase activity is an intrin sic property of secretory immunoglobulin A (sIgA) from milk of healthy human mothers. Polyclonal sIgA was purified by sequential chromatog raphy on protein A-Sepharose, DEAE-cellulose, and gel filtration on Toyopearl HW-55 and Sepharose 4B columns. Its purity was established by one-and two-dimensional SDS-PAGE. The protein kinase activity was inhibited by specific antibodies (Abs) against sIgA, and was stable to acidic and alkaline conditions. Catalytic sIgA showed optimal reaction conditions (pH and MgCl₂ concentration) and substrate specificity dif ferent from those of known protein kinases; i.e., sIgA phosphorylated the serine residues of various milk proteins in the presence of different γ-[³²P]nucleoside-and deoxynucleoside-5’-triphosphates. The homoge neous Fab fragment of sIgA also showed kinase activity. An ATP-binding activity of fractions of sIgA was demonstrated by affinity chromatog raphy on ATP-Sepharose and by covalent binding of an affinity analog of ATP; this activity was mediated by the L chain of sIgA. The authors believe these observations are the first example of the catalytic activity of IgA Abs and of natural catalytic Abs with synthetic activity. In addition, the findings suggest the likelihood that catalytic Abs are generated by the immune system of healthy mothers.     

基于化学蛋白质组学的药物靶标鉴定

通过药物靶标鉴定,可以建立药物活性与细胞表型之间的联系,阐明药物的作用机理,还可以发现药物的脱靶效应和耐药性机制,发现治疗药物的新靶点,并在药物开发的早期阶段预测可能存在的毒性,降低药物研发失败的风险。目前虽然科学技术取得了飞速发展,但是鉴定药物靶标依然是一件令人生畏的工作。本文对近10年来药物靶标鉴定方面的研究进展,特别是无化学标记的新技术进行了评述。     

Quantification of total cholesterol in human milk by gas chromatography

Human milk provides the key nutrients necessary for infant growth and development. The objective of this study was to develop and validate a method to analyze the cholesterol content in liquid human milk samples along lactation. Direct saponification of the sample using ethanolic potassium hydroxide solution under cold conditions was applied and unsaponifiable matter was separated by centrifugation. Cholesterol was converted into its trimethylsilyl ether and the derivative analyzed by gas chromatography coupled with a flame ionization detector. Cholesterol was quantified using epicoprostanol as internal standard. The method is suitable for the determination of cholesterol in only 0.3 g of human milk. It has been validated showing good repeatability (CV(r) < 15%) and intermediate reproducibility (CV(iR) < 15%). The method was used to analyze human milk obtained from five mothers collected at day 30(±3), 60 (±3) and 120 (±3) after delivery. The cholesterol content in human milk slightly decreased from 13.1 mg/100 g at 1 month to 11.3 mg/100 g 120 days after delivery. The method can also be used to determine desmosterol, an intermediate in cholesterol synthesis.     

Proteomics analysis of human breast milk by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with mass spectrometry to assess breast cancer risk

Breast cancer (BC) is one of the most common cancers and one of the most common causes for cancer-related mortality. Discovery of protein biomarkers associated with cancer is considered important for early diagnosis and prediction of the cancer risk. Protein biomarkers could be investigated by large-scale protein investigation or proteomics, using mass spectrometry (MS)-based techniques. Our group applies MS-based proteomics to study the protein pattern in human breast milk from women with BC and controls and investigates the alterations and dysregulations of breast milk proteins in comparison pairs of BC versus control. These dysregulated proteins might be considered potential future biomarkers of BC. Identification of potential biomarkers in breast milk may benefit young women without BC, but who could collect the milk for future assessment of BC risk. Previously we identified several dysregulated proteins in different sets of human breast milk samples from BC patients and controls using gel-based protein separation coupled with MS. Here, we performed 2D-PAGE coupled with nano-liquid chromatography–tandem MS (nanoLC-MS/MS) in a small-scale study on a set of six human breast milk pairs (three BC samples vs. three controls) and we identified several dysregulated proteins that have potential roles in cancer progression and might be considered potential BC biomarkers in the future.     

多维液相色谱分离技术及其在蛋白质组研究中的应用

对近年来多维液相色谱技术及其在蛋白质组学研究中的应用进行了系统综述。详细描述了由不同液相色谱模式构建的多维液相色谱系统,并介绍了其在蛋白质组表达谱、翻译后修饰、定量等方面的应用。此外,还对多维液相色谱的发展趋势和前景进行了展望。     

Characterization of the human serum depletome by label-free shotgun proteomics

While it is known that immunoaffinity depletion of abundant proteins in serum removes additional proteins beyond those targeted, there has been little characterization of the co-depleted proteins in the high abundant fraction, which we refer to here as the "depletome". We present evidence of co-depletion of non-targeted proteins in human serum using a top-20 immunodepletion column, as shown by label-free liquid chromatography mass spectrometry (LC-MS(E)) profiling. This led to identification of 147 proteins which were specific for this fraction and comprised proteins with functions predominantly in binding and transport of nucleotides, metal ions, carbohydrates and lipids. These results suggest that further studies on this commonly ignored serum fraction may provide new insights into clinical proteomics.     

Persistent organochlorines in human breast milk from Al-Sharkia Governorate,Egypt

In the present study, 23 human breast milk samples were collected in January 2009 from Fakous city, Al-Sharkia Governorate, Egypt. The samples were analysed for organochlorine pesticides such as dichlorodiphenyltrichloroethane (DDT) and its metabolites, α, β, and γ-hexachlorocyclohexane (HCH) isomers. The average concentrations of ΣHCHs and ΣDDTs were 225 and 1315?ng?g^?1 lipid respectively. There was no significant difference between the levels of OCP and mother age, while there was a significant difference and correlation between the levels of OCP and the number of times the mother had breast fed (primiparae and multiparae) (p?<?0.05). The results suggested that DDT is still entering the environment depending on the observed ratio of DDE/DDT. The levels of OCP in human milk elucidated that we need to do more regular pollutant monitoring programs.     

Disulfide proteomics for identification of extracellular or secreted proteins

The combination of SDS-PAGE and MS is one of the most powerful and perhaps most frequently used gel-based proteomics approaches in protein identification. However, one drawback of this method is that separation takes place under denaturing and reducing (R) conditions and as a consequence, all proteins with identical apparent molecular mass (Mr) will run together. Therefore, low-abundant proteins may not be easily identified. Another way of investigating proteins by proteomics is by analyzing subproteomes from a total proteome such as phosphoproteomics, glycoproteomics, or disulfide proteomics. Here, we took advantage of the property of secreted proteins to form disulfide bridges and investigated disulfide-linked proteins, using SDS-PAGE under nonreducing (NR) conditions. We separated sera from normal subjects and from patients with various diseases by SDS-PAGE (NR) and (R) conditions, followed by LC-MS/MS analysis. Although we did not see any detectable difference between the sera separated by SDS-PAGE(R), we could easily identify the disulfide-linked proteins separated by SDS-PAGE (NR). LC-MS/MS analysis of the disulfide-linked proteins correctly identified haptoglobin (Hp), a disulfide-linked protein usually found as a heterotetramer or as a disulfide-linked heteropolymer. Western blotting under NR and R conditions using anti-Hp antibodies confirmed the LC-MS/MS experiments and further confirmed that upon reduction, the disulfide-linked Hp heterotetramers and polymers were no longer disulfide-linked polymers. These data suggest that simply by separating samples on SDS-PAGEunder NR conditions, a different, new proteomics subset can be revealed and then identified.     

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。