同系物标记结合凝胶电泳和质谱技术用于蛋白质N端肽鉴定及相对定量

通过酸酐同系物标记蛋白质的N-末端,经过凝胶电泳分离,使用基质辅助激光解析电离飞行时间质谱仪进行鉴定,建立了一种基于化学标记和质谱技术的、用于蛋白质的N-末端鉴定和相对定量新方法。标记后的蛋白质的N端肽以相差14 Da的成对峰形式在质谱中出现,而非N-末端肽则是以单峰呈现,从而实现了末端肽的特异性识别;通过端肽的信号强度对3种标记蛋白质的不同比例的混合溶液实现了相对定量分析。     

修饰组学:解析修饰生物分子功能

基因的时空表达通过转录、翻译和蛋白翻译后修饰这3个层面进行调控.目前在基因组DNA、RNA和蛋白质上发现了丰富的化学修饰.这些化学修饰被认为是调控基因时空表达的另一种新机制.截至目前,在核酸和蛋白质中已经分别发现了超过150和400种不同类型的修饰,阐明这些修饰的生理功能有助于促进对生命体调控机理和运行机制的认识和理解.包括基因组学、转录组学、蛋白质组学和代谢组学在内的组学研究已经蓬勃发展了几十年.鉴于DNA、RNA和蛋白质上含有丰富多样和具有调控作用的化学修饰,本文从修饰和组学的角度提出了新的概念:修饰组学.修饰组学主要是指对DNA修饰、RNA修饰和蛋白质修饰的系统和综合研究.本综述通过介绍DNA、RNA和蛋白质上的化学修饰,总结了它们的生物学功能,探讨了修饰之间的相互作用从而阐释修饰组学,希望为DNA、RNA和蛋白质修饰提供一个系统的蓝图,并促进对其功能的研究.     

蛋白质与线性DNA片断结合过程中的末端效应

各种体外实验技术被广泛地用来研究DNA-蛋白质之间的相互作用, 但体外和体内实验一个最明显的区别是实验使用的DNA片段远远短于基因组DNA, 因而多出了大量线性DNA分子末端. 末端问题曾倍受关注, 若干研究小组在不同系统中对其进行了研究, 但结果却并不一致, 甚至完全相反. 本文利用表面等离子共振技术(SPR)对一系列不同长度非特异DNA和Mnt阻遏蛋白结合和解离过程进行实时监测, 结果表明该蛋白在线性DNA分子末端有比内部位点更高的解离速率. 通过考察在不同位点含有特异序列的DNA与Mnt阻遏蛋白的结合过程, 发现线性DNA分子末端在一定距离内会直接影响DNA与该蛋白的特异性结合.     

丝组二肽——现代蛋白酶分子进化过程中的原始雏形

系统阐述了我们实验室近十五年来对丝组二肽的生物活性的研究.丝组二肽是目前报道的具有多种切割活性的最小活性肽,它不仅能够切割DNA,而且可以切割蛋白质及羧酸酯.丝组二肽是迷你的磷酸酯酶和蛋白水解酶,是现代蛋白酶分子进化过程中的原始雏形.     

基于非特异性蛋白酶连续酶解的蛋白质全序列测定方法

对蛋白质全序列进行测定, 有助于分析蛋白质的结构, 揭示蛋白质的生物学功能. 针对目前基于质谱的蛋白质测序流程中使用特异性蛋白酶酶解产生的肽段种类少、重叠度低、序列拼接困难等问题, 发展了一种基于非特异性蛋白酶连续酶解的蛋白质全序列测定方法. 构建了连续酶解装置, 并使用多种非特异性蛋白酶对蛋白质进行连续酶解. 利用非特异性蛋白酶酶解位点的非特异性、不同的酶解时间以及不同种类蛋白酶酶解产生肽段的互补性, 提高蛋白质酶解肽段的种类和重叠度, 并发展了蛋白质序列拼接算法对液相色谱质谱联用(LC-MS/MS)和从头测序获得的肽段序列进行拼接. 将此方法应用于牛血清白蛋白和单克隆抗体赫赛汀的全序列测定, 在不考虑亮氨酸和异亮氨酸的情况下, 对牛血清白蛋白和赫赛汀轻链的测序准确度达到100%, 赫赛汀重链的测序准确度为99.7%.     

冬小麦麸皮抗冻蛋白一级结构的研究

冬小麦麸皮抗冻蛋白(TaAFP)的一级结构是研究其高级结构和功能的基础.本研究结合N-末端测序技术和肽指纹图谱技术,测定TaAFP的一级结构,并对其同源性进行分析.MALDI-TOF-MS质谱分析显示TaAFP的分子量为13637.711 Da.使用Trypsin(胰蛋白酶)、Hydroxylamine(羟胺)和Chymotrypsin(胰凝乳蛋白酶)分别对TaAFP酶解,并对各个酶解片段的肽指纹图谱进行解析和连接,最终确定TaAFP的一级结构为MARKVIALAFLLLLTISLSKSNAARVKYNGGESGGGGGGGGGGGGGGNGSG-SGSGYGYNYGKGGGQSGGGQGGGGGGGGGGGSNGSGSGSGYGYGYGQGNGGAQGQGSGGGGGGGGGGGGGGSGQGSGSGYGYGYGKGGGGGGGGGGDGGGGGGGGSAYVGRHE,测定覆盖度达到了100%.序列比对分析以及同源性分析结果显示,TaAFP是一种与植物细胞壁和抵抗寒冷相关的蛋白质.     

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。     

应用于天然产物研究与开发的代谢组学数据库

代谢组学在对生物体系的小分子代谢物进行定性和定量研究中产生了大量的数据,成为天然产物研究和开发的重要工具。一系列公开、方便且注释良好的代谢组学数据库和全功能软件的开发促进了天然产物大数据的集成、处理和解释,这不仅有利于数据的存储、管理和分析,还可作为信息共享联动的平台。基于质谱的代谢组学数据库的建立对于天然产物已知成分筛选、未知成分鉴定起着十分重要的作用。对国内外常用的代谢组学数据库及其特征进行综述,强调它们的内容、功能和应用,为数据库的选择提供理论依据,并展望代谢组学数据库的开发趋势。     

超临界二氧化碳介质中钯催化末端炔烃羰基化反应的影响因素

在超临界二氧化碳介质的钯催化末端炔烃羰基化反应中 ,研究了助溶剂、压力和温度等因素对反应的影响。发现末端炔烃羰基化反应生成炔酸酯的最佳条件为 :醇 3～ 4mL ,CO2 压力 7.5MPa和温度 4 0℃。     

必需金属的生物信息学研究现状与展望

金属元素在整个生物界被广泛利用。生物体所必需的金属中除了钾、钙、钠和镁以外均属于微量元素。它们虽然在体内含量很少,但在各种生命活动中发挥着重要的作用。一直以来关于必需金属的研究工作主要集中在金属蛋白和相关代谢机制的实验研究上。随着近年来基因组、蛋白质组等组学数据的不断积累,为开展金属、金属组和金属蛋白质组等相关的生物信息学研究工作提供了重要的条件,让我们可以从系统的角度去进一步认识必需金属的利用、代谢及其生物学功能。本文将分别对若干必需金属(包括铜、钼、镍、钴、锌、铁和硒等)相关的生物信息学研究进展进行介绍,并结合当前离子组学相关的技术方法和研究现状。希望本文有助于我们进一步认识必需金属研究领域的重要问题和未来的发展方向。     

Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins

The new aspect concerning the applicability of histidine and other affinity tags for the purification of oligomeric proteins, with particular emphasis on cleavage efficiency and final yield, is presented in this study. The final yield depends on both the cleavage efficiency and the degree of oligomerization of the protein. Cleavage procedures that are good enough for monomeric proteins can be problematic for oligomeric proteins. Random distribution of uncleaved or partially cleaved affinity tags among oligomers is the main cause of reduced yields. A trimeric protein, tumour necrosis factor alpha (TNF-alpha), bearing different histidine tags, was used as a model protein to explore and confirm this theoretical concept. Analysis of mixed TNF trimers, prepared from tag-free TNF doped with various amounts of histidine-tagged TNF, revealed an increased retention of the trimeric protein on immobilized metal-ion affinity chromatography (IMAC) columns. When 20% of histidine-tagged TNF was added, more than 50% of the protein was retained on the IMAC column. Thus, the applicability of histidine- and other affinity tags for purifying oligomeric proteins is significantly prejudiced in the case of higher oligomers. Various histidine-tags were fused to the N-terminus of full-length TNF-alpha and to the truncated form (dN6) of TNF-alpha. Two-step IMAC separation was used for purification. In the first step, IMAC-1, over 95% purity of histidine-tagged protein was achieved in all cases. Endo- and exoproteolytic removal of histidine tags with enterokinase (EKmax) and aminodipeptidase (DAPase) was studied and the major parameters affecting cleavage efficiency, microheterogeneity and final yield are critically discussed. IMAC-2 was used as the second and final step for removing the cleavage enzyme, cleaved tags, unprocessed protein and some other impurities. Selection of the optimal cleavage enzyme depends on the amino acid composition of the N-terminus and the intended use of the purified protein. The main conclusion is that special caution should be taken when introducing affinity tags to oligomeric proteins, with the final goal to produce pure, tag-free protein with acceptable yields. Given the same enzyme cleavage efficiency one can expect progressively reduced final protein yields with increasing degree of oligomerization. This should be considered as a general rule.     

Optimisation of chemical protein cleavage for erythropoietin semi-synthesis using native chemical ligation

Selective protein cleavage at methionine residues is a useful method for the production of bacterially derived protein fragments containing an N-terminal cysteine residue required for native chemical ligation. Here we describe an optimised procedure for cyanogen bromide-mediated protein cleavage, and ligation of the resulting fragments to afford biologically active proteins.     

Purification of recombinant proteins by chemical removal of the affinity tag

The efficient removal of a N-or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine. Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation. Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification. It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and aPlasmodium falciparum antigen. The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag inEscherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag. Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins.     

Locating apoptosis proteins by incorporating the signal peptide cleavage sites into the general form of Chou's Pseudo amino acid composition

Apoptosis proteins play an essential role in the development and homeostasis of an organism. The accurate prediction of subcellular location for apoptosis proteins is helpful for understanding the mechanism of programmed cell death and their biological functions. In this article, a new apoptosis proteins localization algorithm, named PSSP, is proposed based on the predicted cleavage sites of primary protein sequences. First, protein chains are divided into N‐terminal signal parts and mature protein parts according to their predicted cleavage sites by SignalP. Then, amino acid composition (ACC) of the individual subsequence together with pseudo‐ACC and stereochemical properties of whole chain were extracted to represent a given protein sequence. Jackknife test by support vector machine on three broadly used datasets (ZD98, ZW225, and CL317 datasets) of apoptosis proteins demonstrated that the total accuracies by this approach are 93.9, 87.6, and 91.5%, respectively. In addition, an independent nonapoptosis benchmark dataset (NNPSL) was also used to evaluate the performance of this method, and predictive accuracies for eukaryotic and prokaryotic proteins are also comparable to existing methods. © 2013 Wiley Periodicals, Inc.     

Electrochemical oxidation and cleavage of proteins with on-line mass spectrometric detection: Development of an instrumental alternative to enzymatic protein digestion

An electrochemical flow cell coupled on-line to a mass spectrometer is used to oxidize a range of proteins. Oxidation of tyrosine and tryptophan can give rise to peptide bond cleavage at their C-terminal side. This suggests the possible use of electrochemistry as an alternative protein digestion method. For the small proteins insulin and alpha-lactalbumin (6 and 14 kD) almost all potential sites are cleaved, while for the largest successfully tested protein (carbonic anhydrase, 29 kD) 7 of the 15 available sites were specifically cleaved. Several proteins did not produce peptides upon electrochemical oxidation, possibly due to problems with accessibility of tyrosine and tryptophan residues, or to competing oxidation reactions. Peptides were generally not the major oxidation products: non-cleavage oxidation products observed as protein mass + n x 16 Da, presumably by oxidation of tyrosine, tryptophan, cysteine and methionine, account for the major fraction of protein oxidation products. Nevertheless the amount and variety of cleavage products at the present conditions shows good prospects for further improvement of the system. The efficient protein oxidation also allows the use of the EC-MS system as a tool to study protein oxidation reactions in general. The preconditioning and life history and/or age of the electrochemical cell was relevant to the solvent and sample conditions needed for efficient oxidative cleavage as opposed to other oxidation reactions.     

Photocleavage of lysozyme by cobalt(III) complexes

Photochemical reagents that cleave proteins at specific sites (photoproteases) are useful for studying protein structure and protein-ligand interactions. PolyammineCo(III) complexes are tested here as photochemical probes to cleave proteins. Irradiation of a mixture of lysozyme, a model protein, and polyammineCo(III) complexes resulted in the facile cleavage of the peptide backbone. Photocleavage yielded two fragments of molecular weights 10.6 and 3.7 kDa, and these masses sum to the molecular mass of lysozyme (14.3 kDa). No cleavage was detected in the absence of the metal complex, in the dark, or upon irradiation at wavelengths of >420 nm. The photocleavage yield increased with irradiation time and with the concentrations of the metal complex and the protein. N-terminal sequencing of the 10.6 kDa fragment indicated residues that are identical to the N-terminus of lysozyme, and sequencing of the 3.7 kDa fragment indicated Val-Ala-Trp-Arg, an internal sequence of lysozyme. From the known primary sequence of lysozyme and the sequencing data, the cleavage site was assigned to Trp108-Val109. Molecular modeling indicates that the observed cleavage site is within few angstroms from the proposed metal binding site at Glu35-Asp52. This is the first report of the successful photocleavage of proteins, with high selectivity, by transition metal complexes. This novel observation can facilitate the rational design of transition metal complexes for the photochemical footprinting of metal binding sites on proteins.     

Affinity purification and enzymatic cleavage of inter-alpha inhibitor proteins using antibody and elastase immobilized on CIM monolithic disks

Epoxy-activated monolithic CIM disks seem to be excellent supports for immobilization of protein ligands. The potential use of enzymes, immobilized on monolithic disks for rapid preparative cleavage proteins in solution was investigated. Digestion of complex plasma proteins was demonstrated by using inter-alpha inhibitors with elastase, immobilized on epoxy-activated CIM disks. Recently, a monoclonal antibody against human inter-alpha inhibitor proteins (MAb 69.31) was developed. MAb 69.31 blocks the inhibitory activity of inter-alpha inhibitor proteins to serine proteases. These results suggest that the epitope defined by this antibody is located within or proximal to the active site of the inhibitor molecule. This antibody, immobilized on monolithic disk, was used for very rapid isolation of inter-alpha proteins. The isolated complex protein was used for enzymatic digestion and isolation of cleavage products, especially from inter-alpha inhibitor light chain to elucidate precisely the target sequence for MAb 69.31 by N-terminal amino acid sequencing. Bovine pancreatic elastase immobilized on monolithic disk cleaves inter-alpha inhibitor protein complex into small fragments which are still reactive with MAb 69.31. One of these proteolytic fragments was isolated and partially sequenced. It could be shown that this sequence is located at the beginning of two proteinase inhibitor domains of the inter-alpha inhibitor light chain (bikunin). Elastase immobilized on monolithic disk offers a simple and rapid method for preparative isolation of protease cleavage fragments. The immobilized enzyme is stable and still active after repeated runs. A partial or complete digestion can be achieved by varying the flow rate.     

Coupling caspase cleavage and proteasomal degradation of proteins carrying PEST motif

The degradation is critical to activation and deactivation of regulatory proteins involved in signaling pathways to cell growth, differentiation, stress responses and physiological cell death. Proteins carry domains and sequence motifs that function as prerequisite for their proteolysis by either individual proteases or the 26S multicomplex proteasomes. Two models for entry of substrates into the proteasomes have been considered. In one model, it is proposed that the ubiquitin chain attached to the protein serves as recognition element to drag them into the 19S regulatory particle, which promotes the unfolding required to its access into the 20S catalytic chamber. In second model, it is proposed that an unstructured tail located at amino or carboxyl terminus directly track proteins into the 26S/20S proteasomes. Caspases are cysteinyl aspartate proteases that control diverse signaling pathways, promoting the cleavage at one or two sites of hundreds of structural and regulatory protein substrates. Caspase cleavage sites are commonly found within PEST motifs, which are segments rich in proline (P), glutamic acid (D), aspartic acid (E) and serine (S) or threonine (T) residues. Considering that N- and C- terminal peptide carrying PEST motifs form disordered loops in the globular proteins after caspase cleavage, it is postulated here that these exposed termini serve as unstructured initiation site, coupling caspase cleavage and ubiquitin-proteasome dependent and independent degradation of short-lived proteins. This could explain the inherent susceptibility to proteolysis among proteins containing PEST motif.     

Building and Breaking Bonds via a Compact S‐Propargyl‐Cysteine to Chemically Control Enzymes and Modify Proteins

Analogous to reversible post‐translational protein modifications, the ability to attach and subsequently remove modifications on proteins would be valuable for protein and biological research. Although bioorthogonal functionalities have been developed to conjugate or cleave protein modifications, they are introduced into proteins on separate residues and often with bulky side chains, limiting their use to one type of control and primarily protein surface. Here we achieved dual control on one residue by genetically encoding S‐propargyl‐cysteine (SprC), which has bioorthogonal alkyne and propargyl groups in a compact structure, permitting usage in protein interior in addition to surface. We demonstrated its incorporation at the dimer interface of glutathione transferase for in vivo crosslinking via thiol–yne click chemistry, and at the active site of human rhinovirus 3C protease for masking and then turning on enzyme activity via Pd‐cleavage of SprC into Cys. In addition, we installed biotin onto EGFP via Sonogashira coupling of SprC and then tracelessly removed it via Pd cleavage. SprC is small in size, commercially available, nontoxic, and allows for bond building and breaking on a single residue. Genetically encoded SprC will be valuable for chemically controlling proteins with an essential Cys and for reversible protein modifications.     

Promoting Tag Removal of a MBP-Fused Integral Membrane Protein by TEV Protease

Tag removal is a prerequisite issue for structural and functional analysis of affinity-purified membrane proteins. The present study took a MBP-fused membrane protein, MrpF, as a model to investigate the tag removal by TEV protease. Influences of the linking sequence between TEV cleavage site and MrpF on protein expression and predicted secondary structure were investigated. The steric accessibility of TEV protease to cleavage site of MBP-fused MrpF was explored. It was found that reducing the size of hydrophilic group of detergents and/or extending the linking sequence between cleavage site and target protein can significantly improve the accessibility of the cleavage site and promote tag removal by TEV protease.